Dyes for analysis of protein aggregation

ABSTRACT

Provided are dyes and compositions which are useful in a number of applications, such as the detection and monitoring protein aggregation, kinetic studies of protein aggregation, neurofibrillary plaques analysis, evaluation of protein formulation stability, and analysis of molecular chaperone activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/963,441 filed Apr. 26, 2018 which is a divisional of U.S. patentapplication Ser. No. 15/156,565, filed May 17, 2016, now abandoned,which is a divisional of U.S. patent application Ser. No. 13/510,976,filed Feb. 12, 2013, now abandoned, which is the U.S. national stageapplication of PCT/US2010/03061, filed Nov. 30, 2010, which claimspriority to U.S. patent application Ser. No. 12/592,639, filed Nov. 30,2009 (now U.S. Pat. No. 9,133,343), all of which are hereby incorporatedby reference in its entirety.

SEQUENCE LISTING STATEMENT

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BACKGROUND OF THE INVENTION (1) Field of the Invention

The present application generally relates to dyes and compositionscomprising dyes. More particularly, provided are dyes and compositionsfor identifying and quantifying protein aggregation.

(2) Description of the Related Art

The deposition of insoluble protein aggregates, known as amyloidfibrils, in various tissues and organs is associated with a number ofneurodegenerative diseases, including Alzheimer's, Huntington's andParkinson's diseases, senile systemic amyloidosis and spongiformencephalopathies (Volkova et al., 2007; Stefani & Dobson, 2003).Fibrillar deposits with characteristics of amyloid are also formed byseveral other proteins unrelated to disease, including the whey proteinbeta-lactoglobulin (BLG). All amyloid fibers, independent of the proteinfrom which they were formed, have very similar morphology: long andunbranched, a few nanometers in diameter, and they all exhibit across-beta X-ray diffraction pattern. The ability to form amyloidfibrils of structurally and functionally diverse proteins, some of whichare not associated with amyloid-deposition diseases, suggests that thisproperty is common to all polypeptides Such amyloid structures are alsoknown to possess a binding affinity for certain dyes, notably,thioflavin T and congo red dyes.

Many proteins are known to be only marginally stable in solution,undergoing conformational changes due to various stresses duringpurification, processing and storage (Arakawa et al., 2007). Suchstresses may include elevated temperature, agitation and exposure toextremes of pH, ionic strength, or various interfaces (e.g., anair-liquid interface) and high protein concentration (as observed forsome monoclonal antibody formulations). A wide variety of aggregates areencountered in biopharmaceutical samples, which range in size andphysiochemical characteristics (e.g., solubility, reversibility).Protein aggregates span a broad size range, from small oligomers thatare only a couple nanometers in length to insoluble micron-sizedaggregates that extend to millions of monomeric units. Structurallyaltered proteins have an especially strong tendency to aggregate, oftenleading to their eventual precipitation. Irreversible aggregation is amajor problem for the long-term storage and stability of therapeuticproteins and for their shipment and handling.

Mechanisms of Protein Aggregation

Aggregation is a major degradation pathway that needs to becharacterized and controlled during the development of proteinpharmaceuticals. In the bioprocessing arena, the mechanisms of proteinaggregation are still not fully understood, despite the fact thataggregation is a major problem in therapeutic protein development(Arakawa et al., 2006). One plausible mechanism is that aggregation isdriven or catalyzed by the presence of a small amount of a contaminantwhich serves as a nucleation site. That contaminant could be a damagedform of the protein product itself, host cell proteins, or evennonprotein materials, such as leachates from the container or resinparticles associated with purification of the protein.

If the contaminant is the damaged protein itself, then its aggregationmay lead to soluble oligomers, which become larger aggregates, visibleparticulates, or insoluble precipitates. Such soluble oligomers,host-cell contaminants, or nonprotein materials may serve as a nucleusonto which native proteins assemble and are incorporated into largeraggregates. Damaged forms of a protein product can also arise fromchemical modification (such as oxidation or deamidation) and fromconformationally damaged forms arising from thermal stress, shear, orsurface-induced denaturation. Minimizing protein aggregation thusrequires ensuring both chemical and physical homogeneity; that is,chemically modified or conformationally altered proteins must be removedfrom the final product.

A second mechanism that often leads to protein aggregation is initiatedby the partial unfolding of the native protein during its storage.Protein conformation is not rigid—the structure fluctuates around thetime-averaged native structure to different extents depending uponenvironmental conditions. Some partially or fully unfolded proteinmolecules are always present at equilibrium in all protein solutions,but most such molecules simply refold to their native structure. Theseunfolded proteins may in some instances, however, aggregate with othersuch molecules or may be incorporated into an existing aggregatenucleus, eventually forming larger aggregates, as described above.Factors such as elevated temperature, shaking (shear and air-liquidinterface stress), surface adsorption, and other physical or chemicalstresses may facilitate partial unfolding of proteins, leading to thecascade of events that cause aggregation.

A third aggregation mechanism is reversible self-association of thenative protein to form oligomers. According to the law of mass action,the content of such reversible aggregates will change with total proteinconcentration. The tendency of different proteins to associatereversibly with one another is highly variable, and the strength of thatassociation typically varies significantly with solvent conditions, suchas pH and ionic strength. In principle, these reversible oligomers willdissociate completely as the protein becomes highly diluted, forexample, after delivery of a therapeutic protein in vivo. Consequently,this class of aggregates is generally less of a concern thanirreversible aggregates. Such reversible oligomers can eventually becomeirreversible aggregates, however. Preventing accumulation ofirreversible aggregates may thus require minimizing the reversibleassociation as well. Further, reversible self-association of proteinscan significantly alter overall pharmaceutical properties of productsolutions, such as solution viscosity.

Detection of reversible aggregates can be an especially challengingtask. As such, aggregates can dissociate after their dilution duringattempts to measure them. Additionally, the results of any analysismethod incorporating a separation process in the workflow may dependvery much upon the kinetic rates of the reversibleassociation-dissociation reactions as well as the equilibrium constants.

One consequence of the complexities of monitoring aggregate formationprocesses is the difficulty of linking the effect (presence ofaggregates) to its underlying cause, particularly because the key damagemay occur at a time or place quite separated from the observedconsequence. One example arises during the large-scale production oftherapeutic monoclonal antibodies (MAbs). Acid stability plays a majorrole in the aggregation of MAbs because the process for theirpurification usually involves both low-pH elution from protein-Aaffinity columns and acid-treatment for viral inactivation.

The exposure of MAbs to a low-pH environment can result in small butsignificant conformational changes that can additionally depend uponfactors such as temperature, and solvent composition. While suchpartially unfolded MAbs may not aggregate at low pH, they may aggregateduring subsequent manufacturing steps involving changes in pH or ionicstrength. A larger conformational change at low pH generally leads tomore aggregates upon increasing the pH. Typically, protein aggregateformation from the low-pH structure is not a fast process, but it doesoccur slowly from the association of damaged monomers that have notreturned to their fully native structure. This and other types ofprotein aggregation phenomena may not manifest themselves until monthsafter manufacturing a particular lot of protein or until later stages ofthe product development process. Regardless of the mechanism ofaggregation, preventing aggregation problems requires sensitive andreliable technologies for quantitative determination of aggregatecontent and aggregate characteristics.

Since the earliest clinical applications of protein pharmaceuticals inmedicine, aggregation problems have been implicated in adverse reactionsin humans and other safety issues. In order to minimize such risks fromtherapeutic proteins in the clinic, formulations must be optimized tominimize aggregation during storage, handling, and shipping.

Analysis of Protein Aggregation

The analysis of protein aggregation can be formally classified into fourexperimental types (Arakawa et al., 2006, 2007; Krishnamurthy et al.,2008). The first type of protein aggregation analysis is the mostconventional approach, wherein a small volume of sample is applied to aseparation medium and forms a band or zone. As the band migrates throughthe medium, the proteins separate according to differences in size,electrophoretic charge, or mass. Gel electrophoresis, size exclusionchromatography (SEC), field flow fractionation (FFF), and theoccasionally used band sedimentation technique belong to this class ofmethods. The movement of the band or zone in these methods is oftenmonitored using absorbance or refractive index detection.

In the second type of analysis, the sample initially and uniformly fillsa measurement cell. When an electrical or centrifugal driving force isthen applied, the protein moves along the applied field, leaving aprotein-depleted solvent, which creates a boundary between protein-freeand protein-containing solution phases. The movement of this boundaryover time is measured. This mode of separation is used in analyticalultracentrifugation-sedimentation velocity (AUC-SV) and moving-boundaryelectrophoresis.

The third type of analysis is a measurement of particle size with nophysical separation. An example of this method is referred to ascorrelation spectroscopy and it measures the fluctuation of particles insolution due to Brownian motion (i.e., measures protein diffusioncoefficients). Fluctuations of scattered light and of fluorescenceintensity have been employed in this type of measurement. One of themost widely employed methods in this category is referred to as dynamiclight scattering (DLS).

SEC is the most commonly implemented control method and has become anindustry benchmark for quantification of protein aggregates. SEC is seenas a versatile technique for separation and quantification of proteinaggregates because of its high precision, high throughput, ease of use,compatibility with a quality control (QC) environment, and in most casesability to accurately quantify protein aggregates. In spite of thesestrengths, several concerns exist with the technique including: apotential loss of aggregates (especially multimers), interaction ofsamples with a column matrix, the required change of a sample buffermatrix to an SEC mobile phase, and the inherent requirement for dilutionof samples. Additionally, perturbation of the distribution of proteinaggregates under standard SEC methodological conditions is possible.

AUC-SV relies on hydrodynamic separation of various species in aheterogeneous protein mixture under strong centrifugal force. AUC-SVcomplements SEC in resolving and quantifying low levels of proteinaggregates. The main advantages of AUC-SV are seen in its ability todetect and measure higher order aggregates (which may elute in the voidvolume of an SEC column) and to conduct these measurements withoutexposing samples to a column resin or SEC mobile phase. AUC-SV isconsidered an accurate method because it does not require standards ordissociate aggregates; thus it can be used as an orthogonal method toverify the accuracy of SEC results. AUC-SV suffers from lower precisionthan SEC, however. The practical aspects of AUC-SV that impact precisionand accuracy are beginning to be understood better, and several recentstudies have demonstrated the utility of AUC-SV to detect and quantifyaggregates present at relatively low (˜1%) levels. Despite itsadvantages, AUC-SV is not yet readily amenable for use as a routinerelease test in the biotechnology industry because of issues related tolow throughput, the need for specialized equipment, performance problemsat high protein concentrations, the need for skilled practitioners ofthe method, and difficulty in validating data analysis software.

DLS uses the time-dependent fluctuations of a scattered-light signal tocalculate the hydrodynamic diameter of protein aggregates and theirrelative proportions. This method is highly sensitive to largeaggregates because the intensity of scattered light increasesproportionally with molecular weight. As a result, very large aggregates(e.g., a 1,000-mer) present at trace levels (≤0.1%) can be detected withhigh sensitivity. If present, such aggregates would elute in the voidvolume of an SEC column or they may be filtered out. Although thismethod is ideal for detecting very low mass fractions of largeaggregates, it cannot resolve species that are similar in size. At leasta three- to five-fold difference in hydrodynamic diameter is requiredfor resolving different species. DLS is also not amenable to use as acontrol method because it is semi-quantitative and very sensitive todust or other extraneous particles. Results also depend on the algorithmused for data analysis, which is often proprietary to the manufacturerof a particular instrument.

As an orthogonal technique to SEC and AUC-SV, analytical field-flowfractionation (aFFF) has gained popularity in recent years for itsability to fractionate protein aggregates without a column. aFFF mostcommonly uses two fluid flows (“fields”) in a channel to achieveparticle separation based upon molecular weight and hydrodynamic size(diffusion coefficient). Injected macromolecular species are held inplace by a cross flow on a semi-permeable membrane while a perpendicularchannel flow carries molecules forward based on their diffusioncoefficient, thereby providing size-based fractionation. Because aFFFinvolves no column interactions, it is considered a gentler separationtechnique than SEC. Concerns regarding the interaction of aggregateswith the membrane have yet to be completely addressed, however. aFFF canbe coupled with different detectors including light scattering,refractive index, and ultraviolet (UV) detectors. When compared withSEC, the precision and limit of detection of aFFF is inferior in thehigh-molecular-weight range, because of increased baseline noise.Experimental conditions (e.g., cross-flow rate) for reasonableseparations in one size range are also not generally applicable to othersize ranges, making the technique cumbersome, especially when analyzinga broad range of masses. Along with other limitations, such as the needfor specialized equipment and a skilled operator, and the difficulty invalidating the method prevents the use of aFFF in applications forrelease and stability monitoring.

Resolution and the size range that can be evaluated in one particularanalysis vary widely among the above mentioned techniques. SEC cannothandle a large range of sizes because the pore size or degree ofpolymerization of the resin must be adjusted to the size of the proteinspecies. If a protein sample contains widely different sizes, manytechniques are unsuitable for analyzing all sizes simultaneously. FFFand DLS can cover a very large range of sizes, but in the case of DLS,resolution is generally fairly poor, and FFF entails some trade-offbetween resolution and dynamic range. SV-AUC is intermediate incapability relative to FFF and DLS. The dynamic range of SV-AUC isfairly good, generally a factor of 100 or more in molecular weight atany particular rotor speed. The resolution of SV-AUC is generally notideal for separating monomer from dimer, compared with the best SECcolumns (especially for lower molecular weight proteins). SV-AUC isoften much better, however, than SEC for resolving moderate sizeoligomers, (tetramers to decamers).

The cited analytical techniques also differ significantly with respectto their overall sensitivity, in other words, their ability to detectand quantify small percentages of irreversible aggregates. SEC, FFF, andSV-AUC are all capable of detecting aggregates at levels as low as ˜0.1%when they are well separated from other species. The quantification ofspecies that elute from SEC or FFF is quite good, but aggregates caneasily be lost during the separation process. Thus, SEC and FFF mayprovide good precision but poor accuracy. For SV-AUC, loss of proteinaggregates to surfaces is usually not a problem, but accuratequantification of small oligomers (dimer-tetramer) at total levels of˜2% or less is quite difficult.

The sensitivity of DLS increases linearly with the stoichiometry of theprotein aggregate. DLS is for all practical purposes useless fordetecting oligomers smaller than an octamer, because the techniquecannot resolve such oligomers from monomeric species, and for thoseprotein aggregate species that are resolved, the accuracy of the weightfractions is quite poor, typically plus or minus factors of two to ten.DLS exhibits excellent sensitivity, however, for very large aggregatespecies, which can often be detected at levels far below 0.01% byweight.

Overall, no single analytical technique is ideal for every protein or isoptimal for analyzing the wide range of aggregation problems that canarise with protein pharmaceutical formulation. One important industrytrend are recent requests from regulatory agencies that the proteinaggregation analytical method used for lot release and/or formulationdevelopment. Typically, this means SEC which is cross-checked throughone or more orthogonal approaches to ensure detection of all relevantprotein aggregate species. Comparison of protein aggregate content usingvarious technologies is thus an emerging topic of interest inbiotechnology research.

Fluorescent Dyes and Protein Aggregation

In a fourth method of aggregate analysis, fluorescent dyes have beenused to stain amyloidogenic material in histology, while insights intothe prerequisites and kinetics of amyloid formation have been obtainedby the in vitro analysis of this process using similar dyes (Volkova etal., 2007, 2008; 2009; Demeule et al., 2007). The fluorescent probes,thioflavin T and Congo red, have been the most frequently used dyes todetect the presence of amyloid deposits. Both the benzothiazole dyethioflavin T and the symmetrical sulfonated azo dye congo red have beenadapted to study the formation of amyloid fibrils in solution using thefluorescence properties of these molecules. The amyloid aggregates causelarge enhancements in fluorescence of the dye thioflavin T, exhibitgreen-gold birefringence upon binding the dye congo red, and cause ared-shift in the absorbance spectrum of congo red. Amyloid fibrildetection assays have suffered from several drawbacks, however, whenusing thioflavin T, Congo red and their derivatives. For instance, congored can bind to native α-proteins such as citrate synthase andinterleukin-2 (Khurana et al., 2001). As a consequence of its pooroptical properties, the congo red derivative chrysamine-G only weaklystains neuritic plaques and cerebrovascular amyloid in postmortem tissue(Klunk et al., 1998). Furthermore, the binding of dyes can influence thestability of amyloid aggregates, and the interplay with other components(for example, during testing of potential amyloid inhibitors) isunpredictable (Murakami et al., 2003). Importantly, there exists a greatvariability among the different amyloid fibrils in their ability to bindcongo red and thioflavin T. Fluorescence intensity using thioflavin Tcan vary depending upon the structure and morphology of the amyloidfibrils (Murakami et al., 2003). Despite the widespread use ofthioflavin T, its application to amyloid quantification often generatesinconsistent and inaccurate results. Variations in spectral propertiescaused by buffer conditions and protein-dye ratios result in poorreproducibility, complicating the use of thioflavin T for quantitativeassessment of fibril formation. In the absence of other more reliableassays, investigators have relied heavily upon thioflavin T as areporter probe for amyloid protein aggregation. A reliable method foramyloid quantification likely would be useful not only for detectingmature amyloid fibrils, but also for monitoring the kinetics offibrillogenesis, which is essential for better understanding of theunderlying biophysics and mechanism of the protein aggregation process.Furthermore, such an assay would be a tool for discovery and developmentof therapeutic compounds capable of blocking protein aggregation.

Thus the design of new dyes which can selectively interact withfibrillar amyloidogenic proteins is of substantial importance for basicresearch, and has a crucial practical significance for biotechnology andmedicine. Dialkylamino-substituted monomethine cyanine T-284 andmeso-ethyl-substituted trimethine cyanine SH-516 have demonstratedhigher emission intensity and selectivity to aggregated α-synuclein(ASN) than the classic amyloid stain thioflavin T; while thetrimethinecyanines T-49 and SH-516 exhibit specifically increasedfluorescence in the presence of fibrillar β-lactoglobulin (BLG) (Volkovaet al., 2007). These dyes demonstrated the same or higher emissionintensity and selectivity to aggregated BLG as thioflavin T. Recently,nile red dye has been used to detect antibody A aggregate, but it didnot stain all types of protein aggregates, underscoring the need toseveral analytical methods in order to assess protein aggregation(Demeule et al., 2007).

Optimization of Protein Formulations

Another potential application of a fluorescence based protein aggregatedetection technique relates to pharmaceutical protein formulations (U.S.Pat. Nos. 6,737,401; 5,192,737; 6,685,940; US Patent ApplicationPublication 2008/0125361 A1). The physical stability of pharmaceuticalprotein formulations is of great importance because there is always atime delay between production, protein formulation and its subsequentdelivery to a patient. The physical stability of a protein formulationbecomes even more critical when using drug delivery devices to dispensethe protein formulation, such as infusion pumps and the like. When thedelivery device is worn close to the body or implanted within the body,a patient's own body heat and body motion, plus turbulence generated inthe delivery tubing and pump, impart a high level of thermo-mechanicalstress to a protein formulation. In addition, infusion delivery devicesexpose the protein to hydrophobic interfaces in the delivery syringesand catheters. These interfacial interactions tend to destabilize theprotein formulation by inducing denaturation of the native structure ofthe protein at these hydrophobic interfaces.

In an optimized protein formulation, the protein should remain stablefor several years, maintaining the active conformation, even underunfavorable conditions that may occur during transport or storage.Protein formulation screening needs to be performed before theassessment of safety, toxicity, ADME (absorption distribution metabolismexcretion), pharmacology and the testing of biological activity inanimals. Currently, protein formulation in the pharmaceutical industryis generally a slow process and would benefit from fast formulationscreening approaches that do not require overly complicatedinstrumentation techniques.

The formulation of protein drugs is a difficult and time-consumingprocess, mainly due to the structural complexity of proteins and thevery specific physical and chemical properties they possess. Mostprotein formulations contain excipients which are added to stabilizeprotein structure, such as a particular buffer system, isotonicsubstances, metal ions, preservatives and one or more surfactants, withvarious concentration ranges to be tested. The conventional analyticalmethods usually require a long period of time to perform, typicallytwenty or more days, as well as manual intervention during this period.The development of new formulations is costly in terms of time andresources. Moreover, even for a known protein formulation, batch tobatch quality control analysis is often less than optimal using thecurrent state of the art methods. Therefore, a versatile, reliable,rapid and resource-efficient analytical method is desired for bothdeveloping novel protein formulations and identifying protein stabilityin quality control procedures. The ideal analytical method would besensitive, accurate, and linear over a broad range, resistant tosample-matrix interference, capable of measuring all possible structuralvariants of a protein, and compatible with high throughput screening.

A high throughput screening (HTS) platform for optimization of proteinformulation has been proposed based upon the use of multi-wellmicroplates (Capelle Martinus et al., 2009). Basically, such an HTSplatform was envisioned to consist of two components: (i) samplepreparation and (ii) sample analysis. Sample preparation involvesautomated systems for dispensing the drug and the formulationingredients in both liquid and powder form. The sample analysis involvesspecific methods developed for each protein to investigate physical andchemical properties of the formulations in the microplates.

The techniques that could be coupled with such an HTS platform includeUV-Visible absorbance/turbidity, light scatter, fluorescence intensity,resonance energy transfer, fluorescence anisotropy, Raman spectroscopy,circular dichroism, Fourier transform infrared spectroscopy (FTIR),surface plasmon resonance and fluorescence lifetime. Ideally, however,the analysis technique should be specific, quantitative, robust,cost-effective, easily accessed, easy to use and informative. CapelleMartinus et al. (2009) utilized several assays coupled with HTS tooptimize a salmon calcitonin formulation: turbidity (absorbance at 350nm), intrinsic tyrosine fluorescence, 1-anilino-naphthalene-8-sulfonate(ANS) fluorescence and Nile red fluorescence. Addition of the dyes (Nilered and ANS) were employed to examine protein conformational changes.Their findings were in accordance with the salmon calcitoninformulations that were patented and used commercially, lending credenceto the concept that fluorescent probe-based approaches can be employedin protein formulation optimization activities. The use of severalcomplementary analytical methods permits the selection of formulationsusing carefully designed assay criteria. The investigators found that insome cases, an increase in turbidity was observed without an increase inANS or Nile red fluorescence. In other formulations, an increase influorescence was detected without an increase in turbidity. Thissuggests that these dyes are not necessarily measuring the exact samebiophysical phenomenon as the turbidity measurements. Measuring thefluorescence of at least two dyes in combination with turbidity andintrinsic fluorescence was, therefore, recommended.

Among these techniques, fluorescence detection from externally addeddyes, which enhances fluorescence intensity upon interacting withmisfolded or aggregated protein, is most attractive, because thistechnique requires minimum protein concentration due to its highsensitivity and simple implementation on a microplate reader.

Real time stability testing of a particular formulation may demonstrateno immediately apparent effect on physical or chemical stability.Accelerated stability testing can help, therefore, in facilitating thedetermination of the most suitable excipients and concentrations.Storage at different target temperatures (0-50° C.), illumination ofsamples, mechanical stress (i.e., agitation that simulates handling andtransportation), multiple freeze-thaw cycles (mimicking frozen storage,freeze drying), oxygen purging, increased humidity and seeding aredifferent ways to accelerate protein degradation.

High throughput spectroscopy is a fast and versatile method for initialscreening of the physical stability of protein formulations. Themicroplate well-based platform could be enhanced with accelerated stresstesting and methods to determine chemical stability, e.g.,electrophoresis, HPLC, mass spectrometry. For instance, thioflavin T hasbeen used to select and optimize FDA-approved surfactant(s) in insulinformulations using magnetically stirring to accelerate insulinaggregation (U.S. Pat. No. 6,737,401).

Thermal Shift Assay

Fluorescent dyes have been used to monitor protein stability bysystematically varying the temperature of test samples, also known asthe Thermofluor® technique (U.S. Pat. No. 6,020,141; Matulis et al.,2005; Mezzasalma et al., 2007; Volkova et al., 2008; Ericsson et al.,2006; Todd et al., 2005). Protein stability can be altered by variousadditives including but not limited to excipients, salts, buffers,co-solvents, metal ions, preservatives, surfactants, and ligands.Protein stability can be shifted by various stresses, including elevatedtemperature, referred to as thermal shift, or chemical denaturants, suchas urea, guanidine isocyanate or similar agents. A protein stabilityshift assay offers a wide spectrum of applications in the investigationof protein refolding conditions, optimization of recombinant proteinexpression/purification conditions, protein crystallization conditions,selection of ligand/drug/vaccine/diagnostic reagents and proteinformulations.

The classic thermal shift technology utilizes the dye SYPRO® Orange andinvolves the use of a melting point device to raise the temperaturestepwise (Raibekas, 2008). Thermal shift technology is coupled withaggregation detection technologies, such as light scattering technologyor internal fluorescence from protein (such as tyrosine or tryptophan)to monitor protein aggregation and unfolding respectively. This type oftechnology usually requires a high protein concentration, therefore, itis not cost-effective. In addition, thermal shift technology cannot workeffectively on formulations with low protein concentrations or finalizeprotein formulations which require a very low detection limit (typically˜1-5% protein aggregates).

Fluorometric Screening Assay for Protein Disulfide Isomerase (PDI)

Protein disulfide isomerase (PDI, EC5.3.4.1) is a 57-kDa enzymeexpressed at high levels in the endoplasmic reticulum (ER) of eukaryoticcells (Ferrari and Söling, 1999). PDI was the first enzyme known topossess the disulfide isomerase activity and has been well characterizedover the past three decades. In ER, PDI catalyzes both the oxidation andisomerization of disulfides of nascent polypeptides. Under the reducingcondition of the cytoplasm, endosomes and cell surface, PDI catalyzesthe reduction of protein disulfide bonds.

Folding catalysts such as PDI and peptidylprolyl isomerase accelerateslow chemical steps that accompany folding. Disulfide bond formation canoccur quite rapidly, even before the completion of synthesis, but forsome proteins disulfide bond formation is delayed and occurspost-translationally. PDI catalyzes disulfide formation andrearrangement by thiol/disulfide exchange during protein folding in theER. As a member of the thioredoxin superfamily, which also includeshomologs such as ERp57, PDIp, ERp72, PDIr and ERp5, PDI has twoindependent but non-equivalent active sites, with one positioned closeto the C-terminus and another close to the N-terminus. Each sitepossesses two cysteine residues (CGHC) that cycle between the dithioland disulfide oxidation states. The disulfide bond at the active site ofPDI is a good oxidant that directly introduces a disulfide bond intoprotein substrates. The dithiol redox state is essential for catalyzingdisulfide rearrangements. The necessity of having oxidized and reducedactive sites for catalysis of different steps results in a redoxoptimum. Besides its major role in the processing and maturation ofsecretory proteins in ER, PDI and its homologs have been implicated inother important cellular processes. For example, cellular insulindegradation occurs in a sequential fashion with several identifiedsteps. The initial degradative step occurs in endosomes with two or morecleavages in the B chain occurring. This is followed by reduction ofdisulfide bonds by PDI, or a related enzyme, generating an intact Achain and fragments of B chain. The insulin fragments are furthercleaved by multiple proteolytic systems, such as the lysosomaldegradation pathway.

PDI and its homologs also play roles in the processing and maturation ofvarious secretory and cell surface proteins in the ER following theirsynthesis. Several in vitro studies have also suggested a chaperonefunction of PDI, to assist in protein folding or refolding. During ERstress, as for example during hypoxia in endothelial cells andastrocytes in the cerebral cortex, PDI is up-regulated. This indicatesthat PDI is involved in protecting cells under pathological or stressfulconditions.

Besides ER, PDI also exists on many cell surfaces, such as endothelialcells, platelets, lymphocytes, hepatocytes, pancreatic cells andfibroblasts. For the reductive activity of plasma membrane, PDI isrequired for endocytosis of certain exogenous macromolecules. Thecytotoxicity of diphtheria toxin is blocked by PDI inhibitors, whichblock the cleavage of the inter-chain disulfide bonds in the toxin. PDIalso mediates reduction of disulfide bonds in human immunodeficiencyvirus envelope glycoprotein 120, which is essential for infectivity. PDIinhibitors can thus prevent virus entry into cells. Such functionalactivities make PDI and its homologs attractive drug targets.

Biochemical assays related to measuring PDI activity have beendescribed:

(1) ScRNase assay: PDI converts scrambled (inactive) RNase into native(active) RNase that further acts on its substrate. The reportedsensitivity of the assay is in the micromolar range (Lyles & Gilbert,1991).

(2) The Insulin Turbidity Assay: PDI breaks the two disulfide bondsbetween the two insulin chains (A and B) that results in precipitationof the B chain. This precipitation can be monitored by measuringturbidity (absorbance at 620 nm), which in turn indicates PDI activity.Sensitivity of this assay is in the micromolar range (Lundström &Holmgren, 1990). Recently an end-point, high throughput screening assayof PDI isomerase activity based on enzyme-catalyzed reduction of insulinin the presence of dithiothreitol using hydrogen peroxide as a stopreagent has been developed (Smith et al., 2004; U.S. Pat. No.6,977,142).

(3) The Di-E-GSSG assay: This is the fluorometric assay that can detectpicomolar quantities of PDI and is, therefore, considered the mostsensitive assay to date for detecting PDI activity. Di-E-GSSG has twoeosin molecules attached to oxidized glutathione (GSSG). The proximityof eosin molecules leads to the quenching of its fluorescence. Uponbreakage of the disulfide bond by PDI, however, fluorescence increases70 fold (Raturi & Mutus 2007). Certain common excipients can causesignal generation as well, such as 2-mercaptoethanol and dithiothreitol.

In view of the important functional activities of PDI and homologousenzymes, sensitive, real-time, high throughput methods that are time andcost-effective are highly desirable.

Chaperone/Anti-Chaperone Activity

A chaperone is a protein that can assist unfolded or incorrectly foldedproteins to attain their native state by providing a microenvironment inwhich losses due to competing folding and aggregation reactions arereduced (Puig & Gilbert, 1994). Chaperones also mediate thereversibility of pathways leading to incorrectly folded structures. Oneof the major complications encountered in both in vitro and in vivoprotein folding is aggregation resulting from the commonly encounteredlow solubility of the unfolded protein or different foldingintermediates. The efficiency of folding depends upon how the unfoldedprotein partitions between pathways leading to aggregation and pathwaysleading to the native structure. In vivo, the partitioning betweenproductive and non-productive folding pathways may be influenced by“foldases” and molecular chaperones. Foldases accelerate folding bycatalyzing the slow chemical steps, such as disulfide bond formation andproline isomerization that may retard folding. Molecular chaperones donot appreciably accelerate folding but bind to nonnative proteins in away that is thought to inhibit non-productive aggregation andmisfolding. In order to prevent these improper interactions, chaperonesmust be present at concentrations that are stoichiometric with the newlysynthesized proteins. Consequently, chaperones are often found at veryhigh concentrations in the cell.

PDI is a very abundant protein within cells. Although primarilyclassified as a foldase, PDI has also been shown to possess chaperone oranti-chaperone activity (Puig & Gilbert, 1994). PDI accelerates lysozymefolding, and at high concentration, it displays a chaperone-likeactivity that prevents lysozyme misfolding and aggregation. In addition,PDI also exhibits an unusual “anti-chaperone” activity. Under conditionsthat favor lysozyme aggregation, low concentrations of PDI greatlyreduce the yield of native lysozyme and facilitate the formation ofaggregates that are extensively cross-linked by intermolecular disulfidebonds. Similarly, PDI breaks the two disulfide bonds between two insulinchains (A and B) that results in precipitation of The B chain, thusserving as an “anti-chaperone in this case.” (Lundström & Holmgren.1990.

Alpha-crystallin, a major protein component of the mammalian lens of theeye, belongs to the heat shock protein (Hsp) family and acts as amolecular chaperone by preventing aggregation of target proteins (e.g.beta and gama-crystallins) under stress conditions through the formationof stable, soluble high-molecular mass complexes with them. Aggregationof BLG (beta-lactoglobulin) occurs mainly via intermolecular disulfidebond exchange. Upon heating, BLG aggregates, which can be accelerated bysubjecting the protein to either an elevated pH or through theadditional of DTT. α-crystallin prevents heat-induced BLG aggregation,acting as a chaperone in the absence of DTT; in the presence of DTT,however, this chaperone activity is less efficient due to fasteraggregation of heated and reduced beta-lactoglobulin. Another Hspprotein, Hsp 27, protects myosin 51 from heat-induced aggregation, butnot from thermal denaturation and ATPase inactivation.

Highly sensitive fluorescent probes useful to monitoring various proteinfunctions relating to aggregation should assist in formulationoptimization. Preferably, these probes should be applicable to a broadranges of proteins and concentrations even in the presence ofexcipients, salts and buffers, providing sensitive limits of detectionand excellent linear dynamic ranges.

BRIEF SUMMARY OF THE INVENTION

The present invention provides dyes, reagents and methods useful fordetection of protein aggregates.

In some embodiments, a compound is provided. The compound comprises thestructure

wherein m and n are independently 1, 2 or 3;

wherein L is a linker arm comprising carbon, sulfur, oxygen, nitrogen,or any combination thereof;

wherein R₁, R₂, R₃, R₄, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₉, R₂₀,R₂₁ and R₂₂ are independently hydrogen, halogen, amino, ammonium, nitro,sulfo, sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein thealkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken in combination R₁ and R₂, orR₃ and R₄, or R₉ and R₁₀, or R₁₁ and R₁₂, or R₁₃ and R₁₄, or R₁₅ andR₁₆, or R₁₉ and R₂₀, or R₂₁ and R₂₂ form a five or six membered ringwherein the ring is saturated or unsaturated, substituted orunsubstituted, and wherein R₉ and R₁₀, or R₁₁ and R₁₂, or R₁₃ and R₁₄,or R₁₅ and R₁₆ can comprise alkyl chains that are joined together,wherein a quinoline moiety can be formed;

wherein R₇, R₈, R₁₇ and R₁₈ are independently hydrogen, Z, an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein thealkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken together, R₇ and R₈ and R₁₇and R₁₈, may form a 5 or 6 membered ring wherein the ring is saturatedor unsaturated, substituted or unsubstituted;

-   -   wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester        (COER₂₅), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER₂₅), a        sulfoxide (SOR₂₅), a sulfone (SO₂CR₂₅R₂₆R₂₇), a sulfonamide        (SO2NR₂₅R₂₆), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃        ⁻ER₂₅), a phosphate diester (PO₂ER₂₅ER₂₆), a phosphonate (PO₃ ⁼)        a phosphonate monoester (PO₂ ⁻ER₂₅) a phosphonate diester        (POER₂₅ER₂₆), a thiophosphate (PSO₃ ⁼), a thiophosphate        monoester (PSO₂ ⁻ER₂₅) a thiophosphate diester (PSOER₂₅ER₂₆), a        thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER₂₅)        a thiophosphonate diester (PSER₂₅ER₂₆), a phosphonamide        (PONR₂₅R₂₆NR₂₈R₂₉), its thioanalogue (PSNR₂₅R₂₆NR₂₈R₂₉), a        phosphoramide (PONR₂₅R₂₆NR₂₇NR₂₈R₂₉), its thioanalogue        (PSNR₂₅R₂₆NR₂₇NR₂₈R₂₉), a phosphoramidite (PO₂R₂₅NR₂₈R₂₉) or its        thioanalogue (POSR₂₅NR₂₈R₂₉) where E can be independently O or        S;        -   wherein R₂₅, R₂₆, R₂₇, R₂₈, and R₂₉ are independently a            hydrogen, an unsubstituted straight-chain, branched or            cyclic alkyl, alkenyl or alkynyl group, a substituted            straight-chain, branched or cyclic alkyl, alkenyl or alkynyl            group wherein one or more C, CH or CH₂ groups are            substituted with an O atom, N atom, S atom, or NH group, or            an unsubstituted or substituted aromatic group;    -   wherein Z is attached directly, or indirectly through a second        linker arm comprising carbon, sulfur, oxygen, nitrogen, and any        combinations thereof and wherein the second linker arm may be        saturated or unsaturated, linear or branched, substituted or        unsubstituted or any combinations thereof; and

wherein R₅, R₆, R₂₃ and R₂₄ can independently be hydrogen or an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, or when taken in combination R₅and R₆ or R₂ and R₅ or R₃ and R₆ or R₂₃ and R₂₄ or R₂₂ and R₂₃ or R₂₀and R₂₄ form a five or six membered ring wherein the ring is saturatedor unsaturated, substituted or unsubstituted.

In other embodiments, a compound is provided that exhibits at leastthree times increased fluorescence in the presence of an aggregated formof a protein when compared to the fluorescence exhibited when thecompound is in the presence of the unaggregated form of the protein. Insome embodiments, the compound is D95, D97, L-30, L-33, Lu-1, Lu-2, S-8,S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1,TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135,YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324.

A multi-dye composition comprising at least three dyes is also provided.In this composition, each of the at least three dyes exhibits increasedfluorescence in the presence of an aggregated form of a protein whencompared to the fluorescence exhibited when the compound is in thepresence of the unaggregated form of the protein.

Further provided is a multi-dye composition comprising two or more dyes.In this composition, at least one of the two or more dyes comprises DyeF, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33,S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5,TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135, YAT2148, YAT2149,YAT2150, YAT2213, YAT2214 or YAT2324.

A reactive compound comprising at least one compound from Table 1B orTable 2B is additionally provided. In these embodiments, the compound ismodified by the addition of a reactive group.

Additionally, a labeled target molecule is provided. The labeled targetmolecule comprises a target molecule attached to the above-describedreactive compound through the reactive group.

A solid support attached to the above-described reactive compoundthrough the reactive group is also provided.

A kit for assaying aggregation of a protein is also provided. The kitcomprises in packaged combination: (a) the above-described compound, and(b) instructions for using the compound for assaying aggregation of aprotein.

Another kit for assaying aggregation of a protein is additionallyprovided. The kit comprises in packaged combination: (a) two or morecompounds, wherein each of compound exhibits increased fluorescence inthe presence of an aggregated form of a protein when compared to thefluorescence produced when the compound is in the presence of theunaggregated form of the protein, and (b) instructions therefor.

Additionally provided is a method for detecting an aggregate of aprotein in a sample. The method comprises (a) combining the sample withthe above-described compound or multidye composition; (b) measuring theamount of fluorescence in the mixture;

(c) comparing the amount of fluorescence determined in (b) with theamount of fluorescence in

-   -   (i) a mixture of the compound or multidye composition with a        control sample without aggregated protein, or    -   (ii) a mixture of the compound or multidye composition with a        known standard quantity of aggregated protein; and

(d) determining the aggregation of the protein in the sample based onthe comparison in (c).

A method for separating aggregates of a protein from monomeric forms ofthe protein in a sample is also provided. The method comprises (a)combining the sample to the above-described solid support underconditions where aggregates of the protein preferentially bind to thecompound; and (b) separating sample protein bound to the solid supportfrom unbound protein. In this method, protein bound to the solid supportare substantially aggregates and unbound protein is substantiallymonomers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Panels A-D, are micrographs demonstrating IgG stability in twodifferent buffer formulations.

FIG. 2 is a graph showing the fluorescence of various dye concentrationswith 20 μM of aggregated lysozyme.

FIGS. 3A-B are graphs showing the effect of pH on fluorescent detectionsensitivity and linearity for different probes of the invention.

FIG. 4 is a graph showing the linear dynamic range of lysozyme aggregatedetection using a two dye combination ST (S25 and Tol3) compared withthioflavin T.

FIG. 5 is a graph showing the effective linear dynamic range of antibodyaggregate detection using a two dye combination ST (S25 and Tol3)compared with thioflavin T.

FIGS. 6A-D are graphs showing protein aggregate detection as a functionof various protein species with the dyes S25, Tol3 and thioflavin T.

FIG. 7 is a graph showing the kinetics of lysozyme aggregation monitoredwith dyes S25, Tol3, Thioflavin T and the two dye combination ST (S25and Tol3).

FIG. 8 is a graph showing the kinetics of IgG aggregation as a functionof temperature.

FIG. 9 is a graph showing IgG aggregation induced by temperature (50°C.) as a function of pH.

FIGS. 10A-C are graphs of a high-throughput protein formulationoptimization workflow using IgG and the two dye combination ST (S25 andTol3).

FIG. 11 is a graph showing measurement of the inhibition of Lysozymeaggregation by Chitotriose.

FIG. 12 is a graph showing a thermal shift assay of BLG aggregationusing a dye of the present invention.

FIGS. 13A-B are graphs showing a thermal shift assay of carbonicanhydrase II aggregation at two different pH values using a dye of thepresent invention.

FIGS. 14A-B are graphs comparing the fluorescence response betweenunfolded and aggregated forms of IgG.

FIGS. 15A-C are graphs showing PDI activity monitored by turbidity andby a fluorometric assay using a dye of the present invention.

FIG. 16 is a graph showing activity assay of Hsp 27 (heat shock protein)as a chaperone preventing β-lactoglobulin (BLG) aggregation induced byheat.

FIG. 17 is a graph showing fluorescence of IgG aggregates induced bystirring using a dye combination of the present invention.

FIG. 18, Panels A-B, are fluorescence micrographs of control cells (A)and cells treated with dye YAT2150 (B).

FIG. 19, Panels A-D are fluorescence micrographs of control cells (A)and cells treated with proteasome inhibitors and dye YAT2150.

FIGS. 20A-C are fluorescence micrographs of cells treated with variousdyes to show that dye YAT2150 co-localizes with ubiquitin.

FIG. 21, Panels A-D, are fluorescence micrographs of control cells (A)and cells treated with amyloid beta peptide 1-42 (B, C, D) with (C, D)or without (B) treatment with SMER28, an inducer of autophagy.

FIGS. 22A (Panels A and B) and 22B (Panels A and B) are fluorescencemicrographs of control cells of normal or Alzheimer's disease braintissue after staining with thioflavin T (A) or YAT2150.

FIGS. 23A (Panels A and B) and 23B (Panels A and B) are fluorescencemicrographs showing that dye YAT2150 co-localized with the Tau-13protein in post-mortem brain tissue of Alzheimer's disease patients.

FIGS. 24A-B are graphs comparing YAT2150 (ProteoStat®) withfluorescein-p62 antibody for identifying aggresomes by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

The present invention provides dyes, reagents and methods that areuseful for detecting protein aggregates. In some embodiments, theinvention provides a family of dimeric styryl dyes containing either apicoline or lepidine ring and a dialkyl amino or alkyloxy substituent.The dyes of the invention are useful for generating fluorescence signalsthat depend upon the presence of an aggregated form of a protein, whileconveying minimal levels of signals when only the native form of theprotein is present. A number of novel dimeric styryl dyes having theseproperties are also disclosed. Other dyes have been described previouslyin the context of binding to nucleic acids, but it has been discoveredthat many of these dyes demonstrate a useful property where an enhancedlevel of fluorescence is produced after binding to aggregated forms ofproteins compared to the level that is emitted in the presence of thenative forms. Some of these dyes also exhibit large Stokes shiftsbetween their absorption and emission wavelength optima therebyincreasing the ease of detection.

Thus, in some embodiments, a compound is provided. The compoundcomprises the structure

wherein m and n are independently 1, 2 or 3;

wherein L is a linker arm comprising carbon, sulfur, oxygen, nitrogen,or any combination thereof;

wherein R₁, R₂, R₃, R₄, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₉, R₂₀,R₂₁ and R₂₂ are independently hydrogen, halogen, amino, ammonium, nitro,sulfo, sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein thealkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken in combination R₁ and R₂, orR₃ and R₄, or R₉ and R₁₀, or R₁₁ and R₁₂, or R₁₃ and R₁₄, or R₁₅ andR₁₆, or R₁₉ and R₂₀, or R₂₁ and R₂₂ form a five or six membered ringwherein the ring is saturated or unsaturated, substituted orunsubstituted, and wherein R₉ and R₁₀, or R₁₁ and R₁₂, or R₁₃ and R₁₄,or R₁₅ and R₁₆ can comprise alkyl chains that are joined together,wherein a quinoline moiety can be formed;

wherein R₇, R₈, R₁₇ and R₁₈ are independently hydrogen, Z, an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein thealkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken together, R₇ and R₈ and R₁₇and R₁₈, may form a 5 or 6 membered ring wherein the ring is saturatedor unsaturated, substituted or unsubstituted;

-   -   wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonate ester        (COER₂₅), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER₂₅), a        sulfoxide (SOR₂₅), a sulfone (SO₂CR₂₅R₂₆R₂₇), a sulfonamide        (SO2NR₂₅R₂₆), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃        ⁻ER₂₅), a phosphate diester (PO₂ER₂₅ER₂₆), a phosphonate (PO₃ ⁼)        a phosphonate monoester (PO₂ ⁻ER₂₅) a phosphonate diester        (POER₂₅ER₂₆), a thiophosphate (PSO₃ ⁼), a thiophosphate        monoester (PSO₂ ⁻ER₂₅) a thiophosphate diester (PSOER₂₅ER₂₆), a        thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO⁻ER₂₅)        a thiophosphonate diester (PSER₂₅ER₂₆), a phosphonamide        (PONR₂₅R₂₆NR₂₈R₂₉), its thioanalogue (PSNR₂₅R₂₆NR₂₈R₂₉), a        phosphoramide (PONR₂₅R₂₆NR₂₇NR₂₈R₂₉), its thioanalogue        (PSNR₂₅R₂₆NR₂₇NR₂₈R₂₉), a phosphoramidite (PO₂R₂₅NR₂₈R₂₉) or its        thioanalogue (POSR₂₅NR₂₈R₂₉) where E can be independently O or        S;        -   wherein R₂₅, R₂₆, R₂₇, R₂₈, and R₂₉ are independently a            hydrogen, an unsubstituted straight-chain, branched or            cyclic alkyl, alkenyl or alkynyl group, a substituted            straight-chain, branched or cyclic alkyl, alkenyl or alkynyl            group wherein one or more C, CH or CH₂ groups are            substituted with an O atom, N atom, S atom, or NH group, or            an unsubstituted or substituted aromatic group;    -   wherein Z is attached directly, or indirectly through a second        linker arm comprising carbon, sulfur, oxygen, nitrogen, and any        combinations thereof and wherein the second linker arm may be        saturated or unsaturated, linear or branched, substituted or        unsubstituted or any combinations thereof; and

wherein R₅, R₆, R₂₃ and R₂₄ can independently be hydrogen or an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, or when taken in combination R₅and R₆ or R₂ and R₅ or R₃ and R₆ or R₂₃ and R₂₄ or R₂₂ and R₂₃ or R₂₀and R₂₄ form a five or six membered ring wherein the ring is saturatedor unsaturated, substituted or unsubstituted.

In many of these embodiments, the compound exhibits increasedfluorescence in the presence of an aggregated form of a protein whencompared to the fluorescence exhibited when the compound is in thepresence of the unaggregated form of the protein.

These compounds can be modified by the addition of charged groups, asexemplified by sulfonates, phosphates, phosphonates and theirderivatives and/or polar groups as exemplified by sulfoxide, sulfone andsulfonamide moieties.

It is also understood that when a dye comprises an anionic group, therewill also be a cationic counterion present. Any cation may serve thispurpose as long as it does not interfere with the use of the dye.Examples of cations that may serve as counterions can include but arenot limited to hydrogen, sodium, potassium, lithium, calcium, cesium,ammonium, alkyl ammonium, alkoxy ammonium and pyridinium. It is alsounderstood that when a dye comprises a cationic group, there will alsobe an anionic counterion present. Any anion may serve this purpose aslong as it doesn't interfere with the use of the dye. Examples of anionsthat may serve as counterions can include but not be limited toperchlorate (ClO₄ ⁻), sulfate (SO₄ ⁼), sulfonate, alkane sulfonate, arylsulfonate, phosphate, tosylate, mesylate and tetrafluoroborate moietiesand halides such as a bromide, chloride, fluoride and iodide. In somecases the counterion or counterions are provided by the dye being a saltwhere they exist as separate ionic species. In other cases, thecounterion or counterions may be present as part of the compound(sometimes called inner salts). It is understood that there may also bea combination of ions that are provided by the compound and salts. Withregard to acid moieties that are shown in forms such as COOH it is alsounderstood that these compounds may be found in ionized forms such asCOO⁻.

It should also be appreciated by those skilled in the art that thestoichiometric number of counterion or counterions which balance thecharge or charges on the compound can be the same or they can bedifferent provided that the counterions balance the charge(s) on thecompound. The combination of counterions can be selected from any of theabove mentioned anions. This applies for the combination of cationsalso.

It should be further appreciated by those skilled in the art that theforegoing descriptions of the anions and their stoichiometric numberand/or combination are applicable to the compounds and dyes of thepresent invention, and to methods which use these compounds and dyes.

Alkyl or alkoxy R groups in the above compounds may be substituted orunsubstituted. Examples of substitutions can include but are not limitedto one or more fluorine, chlorine, bromine, iodine, hydroxy, carboxy,carbonyl, amino, cyano, nitro or azido groups as well as other alkyl oralkoxy groups. The length of the alkoxy groups may be as desired. Forinstance, they may independently comprise from 1 to 18 carbons inlength. They may be shorter as well, for instance they may be only 1 to6 carbons in length in a dye molecule of the present invention.

The polar groups, charged groups and other substituents may be connectedto the dye directly or they may be connected by a linker arm comprisingcarbon, nitrogen, sulfur, oxygen or any combination thereof. The linkerarm may be saturated or unsaturated, linear or branched, substituted orunsubstituted as well as any combination of the foregoing.

As described above some of the R groups may be joined together to formone or more fused 5 or 6 membered ring structures. It is understood thatthe complex rings that are formed by closure of R groups may be furthersubstituted with any of the R groups described previously. Examples ofcomplex rings that may be formed for the picoline or lepidine portion ofthe cyanine dyes of the invention can comprise but not be limited to:

Examples of rings and complex rings that may be part of the styrylportion of the dye can comprise but not be limited to:

In various embodiments, the compound comprises the structure

In some of these embodiments, each of R₅, R₆, R₂₃ and R₂₄ are a methylor an ethyl moiety.

As described in Example 1, numerous compounds having the abovestructure, as well as other compounds, were tested for the ability toexhibit increased fluorescence in the presence of an aggregated form ofa protein (human α-synuclein) when compared to the fluorescenceexhibited when the compound is in the presence of the unaggregated formof the protein. The excitation and emission wavelength in the presenceand absence of the protein aggregate was also determined. Results ofthese tests, and the structures of the tested compounds, are provided inTables 1 and 2. Table 1 gives results where the compounds exhibited aratio of 3 or more for fluorescence from binding to protein aggregatescompared to being in the presence of monomeric protein; Table 2 givesresults with other compounds.

TABLE 1 Compounds tested that exhibit a ratio of 3 or more forfluorescence from binding to protein aggregates compared to being in thepresence of monomeric protein. A. Properties of compounds. Dye Dye Dyewith Dye with Alone: Alone: Aggregate: Aggregate: Fluore- Fluore-Fluore- λ_(Ex) λ_(Em) λ_(Ex) λ_(Em) cence cence cence I_(Agg)/ Dye (nm)(nm) (nm) (nm) I_(dye) I_(Mono) I_(Agg) I_(Mono) S25 485 613 516 607 3.44.6 87.3 19.0 S43 527 637 550 623 0.35 0.58 27.3 47 TOL3 471 611 511 6032.7 2.7 40.2 14.9 Yat 500 620 535 613 4.2 4.9 63.2 12.9 2134 Yat 520 632553 625 1.2 3.4 53 15.6 2148 Yat 502 614 534 617 0.6 0.7 29.5 42 2149Yat 485 612 515 610 6.7 9.7 42.3 4.4 2150 F 460 610 518 607 3.6 3.4 57.416.9 L-33 465 527 462 504 7.7 7.6 53 7.0 S49 501 584 524 576 2.2 2.220.1 9.1 S33 479 616 513 611 5.5 5.7 19.4 3.4 TOL- 389 539 554 603 106.5 22.5 3.5 11 SL- 491 578 516 578 7.4 7.5 30.3 4.1 2131 SL- 401 608400 608 7.5 8 25.4 7.5 2592 Tio-1 494 578 526 578 2.8 2.8 19 6.8 S-13568 662 580 670 0.2 0.2 3.1 15.5 L-30 457 515 478 512 1.5 1.8 8 4.4 YA-1446 491 461 498 7.7 11 45 4.1 YA-3 460 514 456 537 6.4 7.9 24.2 3.1(Diph40) TOL-2 527 595 566 600 0.5 0.5 3.5 7 [T-33] TOL-5 428 581 460535 3.8 4.5 22.2 4.9 Dil-10 548 595 564 599 3.7 3.6 15.3 4.3 [TOL-7]S-39 540 599 577 605 3.1 3.3 11.3 3.4 Fm [b] 461 610 504 597 5.5 5.5 213.8 S-42 547 600 559 603 1.1 1.1 5 4.5 S-48 491 581 527 588 2.3 2.3 15.86.9 TOL-6 501 559 512 559 4.6 4.6 17.5 3.8 Lu-1 453 583 452 526 7.3 7.624 3.2 Lu-2 473 506 485 503 3.8 3 14 4.7 Yat2135 500 618 540 620 0.9 0.712.5 17.9 Yat2214 507 626 549 625 1.5 1.4 6.4 4.6 Yat2213 483 622 540622 0.6 1.2 5.5 4.6 D-95 450 585 555 598 0.6 1 8.4 8.4 D-97 516 650 587650 3.6 3.7 13.4 3.6 S-8 547 671 566 667 0.8 0.8 2.8 3.5 Yat2324 500 619551 619 0.8 0.7 7.1 10.1 S-22 543 598 562 602 0.5 0.8 2.4 3.0 B.Structures of compounds. Dye Structure S25

S43

TOL3

Yat 2134

Yat 2148

Yat 2149

Yat 2150

F

L-33

S49

S-33

TOL-11

S-22

SL-2131

SL-2592

Tio-1

S-13

L-30

YA-1

YA-3 (Diph40)

TOL-2 [T-33]

TOL-5

Dil-10 [TOL-7]

S-39

Fm [b]

S-42

S-48

TOL-6

Lu-1

Lu-2

Yat-2135

Yat-2214

Yat-2213

D-95

D-97

S-8

Yat2324

TABLE 2 Compounds tested that exhibit a ratio of less than 3 or more forfluorescence from binding to protein aggregates compared to being in thepresence of monomeric protein. A. Properties of compounds Dye with Dyewith Dye alone: Dye alone: aggregate: aggregate: Fluorescence ExcitationEmission Excitation Emission enhancement: wavelength wavelengthwavelength wavelength Aggregate/ Dye (nm) (nm) (nm) (nm) monomer S-11531 594 560 600 2.6 S-12 539 597 553 599 2.2 SH-330 393 278 398 483 1.6SH-654 370 443 359 434 0.91 SH-675 445 472 449 475 1.8 SH-975 471 631471 630 1.8 SH-1036 478 611 464 605 2.7 Sl-2599 468 564 468 569 1.1Sl-2600 518 535 518 536 1.9 S-7 460 612 465 609 1.1 L-28 460 654 572 5771.4 L-31 450 527 462 534 2.7 TOL-4 488 665 458 654 1.4 TOL-10 394 544397 539 1.4 S-26 532 593 562 602 2.7 S-29 543 597 554 600 1.6 S-44 498586 525 582 2.8 S-45 534 596 558 600 2.3 Dbt-5 539 597 545 598 2.2[TOL-9] S-30 530 598 570 600 1.9 Sip-7 397 576 397 576 1.2 [TOL-12] S-28384 608 384 608 1 S-23 464 546 471 553 1.2 SH-1070 408 500 408 480 1.2Yat2212 485 623 530 620 2.6 D-91 395 520 396 517 1.03 D-78 426 621 426621 0.94 D-68 553 696 558 694 1.1 D-69 483 638 483 637 1.07 D-160 481631 493 617 1.2 D-155 500 625 516 619 1.6 D-72 380 477 375 469 1.1 D-163493 588 490 589 1.06 D-159 487 603 494 593 1.5 D-80 489 669 486 668 0.31D-84 494 623 507 602 2.8 D-90 475 662 479 655 0.36 D-162 472 706 565 6929.9 D-70 506 615 506 614 0.92 D-86 388 544 387 544 1 D-87 430 534 428534 1.08 D-85 450 515 534 608 2.3 Tol-24 530 594 540 598 2.4 Yat2325 503624 538 622 2.5 S-5 527 597 535 598 1.1 S-38 535 599 555 602 2.1 S-37545 600 551 602 1.9 S-3 562 595 565 596 1.2 S-27 522 607 540 608 1.2SIP-2 397 576 397 575 1.3 D-74 517 601 527 601 1 Sbt 520 592 551 594 2.8D-75 494 554 494 555 1.5 D-71 482 585 498 587 1.4 Dbo-10 505 559 515 5972.1 SI-1999 582 595 582 595 1 SL-42 555 567 555 567 0.9 Dimer- 431 577440 580 1.12 NN SIP-3 398 579 408 582 0.98 SIP-10 404 582 440 590 0.79Dst-NN-6 397 572 402 572 1 SIP-8 446 582 442 584 0.28 Dst-NN- 396 572398 572 1.24 10 Dst-NN- 398 584 413 600 0.76 11 Dst-NN- 404 581 412 5950.79 12 SI-1035 512 545 512 546 1 SI-1047 574 596 575 596 0.95 SI-1056546 571 548 572 1 SL-1722 673 700 676 699 1 SL-2153 547 573 547 573 0.9SI-2596 491 594 492 609 1.1 SI-2611 456 554 460 555 1 T-164 559 575 559572 1 SH-0229 520 628 525 641 0.7 T-33 589 656 589 656 0.7 SH-0423 409536 409 588 1 SH-0428 588 601 588 603 2.3 SH-0627 558 569 558 569 1.1T-333 559 576 559 576 1 T-74 561 576 561 576 0.9 SH-0999 640 653 585 5962 T-165 583 623 588 632 2.3 T-364 582 628 581 630 1.4 Dst-NN- 398 576409 576 0.65 13 T-119 530 635 532 636 1 T-15 554 571 564 575 1.2 TOL-26563 609 564 607 0.9 Dst-NN-8 366 474 374 472 1.26 SL-2057 589 603 591605 1.26 SL-2059 582 608 582 607 0.97 SL-2132 532 604 558 609 1.46 B.Structures of compounds. Dye Structure S-11

S-12

SH-330

SH-654

SH-675

SH-975

SH-1036

SI-2599

SI-2600

S-7

L-28

L-31

L-28

TOL-4

TOL-10

S-26

S-29

S-44

S-45

S-30

Dbt-5 [TOL-9]

Sip-7 [TOL-12]

S-28

S-23

SH-1070

Yat-2212

D-91

D-78

D-68

D-69

D-160

D-155

D-72

D-163

D-159

D-80

D-84

D-90

D-162

D-70

D-86

D-87

D-85

Tol-24

Yat2325

S-5

S-38

S-37

S-3

S-27

SIP-2

D-74

Sbt

D-75

D-71

Dbo-10

SI-1999

SL-42

Dimer-NN

SIP-3

SIP-10

Dst-NN-6

SIP-8

Dst-NN-10

Dst-NN-11

Dst-NN-12

SI-1035

SI-1047

SI-1056

SL-1722

SL-2153

SI-2596

SI-2611

T-164

SH-0229

T-33

SH-0423

SH-0428

SH-0627

T-333

T-74

SH-0999

T-165

T-364

Dst-NN-13

T-119

T-15

TOL-26

Dst-NN-8

SL-2057

SL-2059

SL-2132

Although the compounds in Tables 1 and 2 are shown with a particularcounterion, it should be understood that the compounds can also utilizeother counterions as described above. As such, when the above compoundsare identified by name herein, the named compound includes the structureidentified in Table 1 or 2 with any counterion, unless the counterion isparticularly specified.

Notable examples of compounds useful compounds from Table 1 include

wherein X comprises an anion (compound S25).

wherein X comprises an anion (TOL3).

wherein X comprises an anion (S43).

wherein X comprises an anion (YAT2134).

wherein X comprises an anion (YAT2148).

wherein X comprises an anion (YAT2149).

wherein X comprises an anion (YAT2150).

Especially useful for many purposes are dyes that have fluorescenceemissions in the range of 600-650 nM since such dyes can avoidinterference of biological proteins for the application in tissuestaining, such as green fluorescent proteins (GFPs). Excitationfluorescence for such dyes are preferred to be in the range of 500-600nM. It can be seen that the dyes in Table 1 fulfill these requirementswhere the maxima of the fluorescence excitation spectra of these dyes inthe presence of aggregates of α-synuclein (ASN) are between 511 and 553nm, and fluorescence emission have their maxima between 603 and 625 nm.The values of the fluorescence quantum yield (QY) of the dyes of theinvention in the presence of saturating concentrations of fibrillarprotein are situated in the range between 0.01 and 0.08, which allowusing relatively small amounts of dye for interaction with proteinaggregates, tissues or cell staining. Stokes shift of the dyes of theinvention are in the range of 73 to 95 nm and are much larger than theclassic amyloid detection dyes, such as thioflavin T, which only has a23 nm Stokes shift. The wider Stokes shift of the dyes of the presentinvention ensures a much lower overlap between excitation and emission,thus allowing more flexible filter set selection, such as a wideexcitation and or emission filter to improve the brightness of the dyeor increasing the exposure time to enhance the fluorescence intensity.With these considerations, particularly useful compounds from Table 1include S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135 andYAT2324.

It is to be understood that with any particular dye, the excitationmaximum, emission maximum, and/or ratio of fluorescence intensity in thepresence of aggregates vs. monomers can vary to some extent withdifferent proteins. Thus, the selection of a dye to use for detection ofthe aggregates of any particular protein could benefit from informationof the fluorescence characteristics of the dye with the particularprotein. Such information can be obtained for any protein-dyecombination without undue experimentation, for example by using themethods described in Example 1. Nonlimiting examples of useful proteinswhose aggregation could be detected using the above compounds includeimmunoglobulin, a DNA polymerase or a fragment thereof, α-synuclein,synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutant of CFTR,amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1,ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor,p62/sequestosomel (SQSTM1), Pael receptor, serum amyloid A,transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrialnatriuretic factor, lysozyme, insulin, fibrinogen, crystallin,surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin,neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin,lactadherin, p53, procalcitonin, cytokeratin, GFAP, ATP7B, prolylhydroxylase PHD3, presenilin, or huntingtin.

A further consideration of the present invention, is that detectionand/or quantification of aggregates may also be improved by a mixture ofdyes where at least one of the dyes is one of the compounds illustratedin Table 1. The additional dye or (dyes) may also be from Table 1 or 2.The use of more than one dye may widen the breadth of proteins that willsuccessfully generate signals after aggregation when these dyes becomebound. The signal will derive from the net amount of fluorescenceenhancement derived from each dye in the mixture. Particularly usefulmulti-dye compositions comprise dyes where the emission maximum of eachdye is within 150 nm of the emission maximum of each of the other dyes.For some applications, multi-dye compositions may be even more usefulwhere the compositions comprise dyes where the emission maximum of eachdye is within 50 nm of the emission maximum of each of the other dyes.

Thus, in some embodiments, a multi-dye composition is provided. Thismulti-dye composition comprises at least three dyes, where each of theat least three dyes exhibits increased fluorescence in the presence ofan aggregated form of a protein when compared to the fluorescenceexhibited when the compound is in the presence of the unaggregated formof the protein. In some of these embodiments, each of the three dyes isselected from the group consisting of Dye F, Dye Fm(b), D95, D97, L-30,L-33, Lu-1, Lu-2, S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49,SL2131, SL2592, Tio-1, TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1,YA-3, YAT2134, YAT2135, YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 andYAT2324. In other embodiments, at least one of the three dyes isselected from the group consisting of S25, S43, TOL3, YAT2134, YAT2148,YAT2149, S13, YAT2135, YAT2324 and YAT2150.

Another multi-dye composition is also provided herein. This multi-dyecomposition comprises two or more dyes, where at least one of the two ormore dyes comprises Dye F, Dye Fm(b), D95, D97, L-30, L-33, Lu-1, Lu-2,S-8, S13. S22, S25, S33, S39, S42, S43, S48, S49, SL2131, SL2592, Tio-1,TOL-2, TOL-3, TOL-5, TOL-6, TOL-7, TOL-11, YA-1, YA-3, YAT2134, YAT2135,YAT2148, YAT2149, YAT2150, YAT2213, YAT2214 or YAT2324. In some of theseembodiments, at least one of the two dyes is selected from the groupconsisting of S25, S43, TOL3, YAT2134, YAT2148, YAT2149, S13, YAT2135,YAT2324 and YAT2150. In other embodiments, both of the two dyes areselected from the group consisting of S25, S43, TOL3, YAT2134, YAT2148,YAT2149, S13, YAT2135, YAT2324 and YAT2150. In particular embodiments,the two dyes are S25 and TOL3. See, e.g., Example 26.

In another embodiment of the present invention, any of the above dyesfurther comprises a reactive group, thereby allowing their attachment totargets of interest. Examples of reactive groups that may find use inthe present invention can include but not be limited to a nucleophilicreactive group, an electrophilic reactive group, a terminal alkene, aterminal alkyne, a platinum coordinate group or an alkylating agent.

There are a number of different electrophilic reactive groups that mayfind use with the present invention; examples can include but not belimited to isocyanate, isothiocyanate, monochlorotriazine,dichlorotriazine, 4,6,-dichloro-1,3,5-triazines, mono- or di-halogensubstituted pyridine, mono- or di-halogen substituted diazine,maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide,glyoxal and aldehyde groups. Nucleophilic reactive groups can includebut not be limited to reactive thiol, amine and hydroxyl groups. Forpurposes of synthesis of dyes, reactive thiol, amine or hydroxyl groupscan be protected during various synthetic steps and the reactive groupsgenerated after removal of the protective group. Use of a terminalalkene or alkyne groups for attachment of markers has been previouslydescribed in U.S. Patent Application Serial No. 2003/0225247, herebyincorporated by reference. The use of platinum coordinate groups forattachment of other dyes has been previously disclosed in U.S. Pat. No.5,580,990 and the use of alkyl groups has been previously described inU.S. Pat. No. 6,593,465 B1, both of which patents are herebyincorporated by reference. In some cases the molecules that have beendisclosed already have a suitable group that can be used as a reactivegroup; in other cases standard chemical manipulations can be used tomodify a dye to comprise a desired reactive group.

Thus, the present invention provides a composition comprising any of theabove-identified compounds, where such compound or compounds have beenmodified by the addition of a reactive group (Rx) for attachment of atarget molecule thereto. The reactive group (Rx) comprises anelectrophilic reactive group comprising isocyanate, isothiocyanate,monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1,3,5-triazines,mono- or di-halogen substituted pyridine, mono- or di-halogensubstituted diazine, maleimide, haloacetamide, aziridine, sulfonylhalide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimideester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyldithio)-propionamide, glyoxal or aldehyde groups, and a combination ofany of the foregoing. In another embodiment, the reactive group (Rx)comprises a nucleophilic reactive group comprising reactive thiol, amineor hydroxyl, and a combination of the foregoing. In other aspects, thereactive group (Rx) comprises a terminal alkene group, a terminal alkynegroup, a nickel coordinate group or a platinum coordinate group forattachment. The reactive group (Rx) can be attached to the compoundthrough a linker arm.

Another aspect of the present invention is a labeled target moleculecomprising a target molecule attached to any of the above-describedreactive compounds through the reactive group. The target molecule isnot narrowly limited to any particular type of molecule, and cancomprise any molecule that can be attached to the above-describedreactive compounds. Nonlimiting examples of target molecules include anucleoside, a nucleotide, an oligonucleotide, a polynucleotide, apeptide nucleic acid, a protein, a peptide, an enzyme, an antigen, anantibody, a hormone, a hormone receptor, a cellular receptor, alymphokine, a cytokine, a hapten, a lectin, avidin, streptavidin,digoxigenin, a carbohydrate, an oligosaccharide, a polysaccharide, alipid, a liposomes, a glycolipid, a viral particle, a viral component, abacterial cell, a bacterial component, a eukaryotic cell, a eukaryoticcell component, a natural drug or synthetic drug, and any combinationthereof.

Examples of useful target molecules and solid-phase supports can includebut are not limited to a nucleoside, nucleotide, oligonucleotide,polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen,antibody, hormone, hormone receptor, cellular receptor, lymphokine,cytokine, hapten, lectin, avidin, streptavidin, digoxigenin,carbohydrate, oligosaccharide, polysaccharide, lipid, liposomes,glycolipid, viral particle, viral component, bacterial cell, bacterialcomponent, eukaryotic cell, eukaryotic cell component, natural drug,synthetic drug, glass particle, glass surface, natural polymers,synthetic polymers, plastic particle, plastic surface, silicaceousparticle, silicaceous surface, organic molecule, dyes and derivativesthereof.

The nucleoside, nucleotide, oligonucleotide, or polynucleotide cancomprise one or more ribonucleoside moieties, ribonucleotide moieties,deoxyribonucleoside moieties, deoxyribonucleotide moieties, modifiedribonucleosides, modified ribonucleotides, modifieddeoxyribonucleosides, modified deoxyribonucleotides, ribonucleotideanalogues, deoxyribonucleotide analogues or any combination thereof.

As indicated above, the target molecule of these embodiments may havedyes as targets thereby creating composite dyes. By joining the dyes ofthe present invention to another dye, unique properties may be enjoyedthat are not present in either dye alone. For instance, if one of thedyes of the present invention is joined to another dye such that itcreates an extended conjugation system, the spectral characteristics ofthe dye may be different than either dye component.

Another example of this method is where the conjugation systems do notoverlap but the proximity allows an internal energy transfer to takeplace thereby extending the Stokes shift, a system that is commonlyreferred to as FRET (Fluorescent Resonance Energy Transfer) or EnergyTransfer in short. For an example of this, see U.S. Pat. Nos. 5,401,847;6,008,373; 5,800,996, all three of which are hereby incorporated byreference.

Other properties may also be enhanced by this joining; for example, ithas been previously described that the joining together of two ethidiumbromide molecules generates a dye that has enhanced binding to nucleicacids and novel fluorescent properties that are different from themonomeric forms (U.S. Patent Application Publication No. 2003/0225247,hereby incorporated by reference). Other composite dyes have beendescribed that simultaneously enjoy both properties, i.e., enhancedbinding and energy transfer (U.S. Pat. No. 5,646,264, herebyincorporated by reference). Furthermore, these composites dyes are notlimited to binary constructs of only two dyes, but may compriseoligomeric or polymeric dyes. These composite dyes may be comprised ofthe same dye or different dyes may be joined together depending upon theproperties desired.

Utility may also be achieved by attaching a dye of the present inventionto a target specific moiety. Thus, binding between the target specificmoiety and its corresponding target may be monitored by essentiallydetermining the presence or amount of dye that is bound to the target.Well-known examples of such assays are hybridizations betweencomplementary nucleic acids as well as binding that take place betweenantibodies and their corresponding antigens.

Other binding pairs that may be of interest can include but not belimited to ligand/receptor, hormone/hormone receptor,carbohydrate/lectin and enzyme/substrate. Assays may be carried outwhere one component is fixed to a solid-phase support and acorresponding partner is in solution. By binding to the component fixedto the support, the partner now becomes attached to the support as well.A well-known example of this method is the microarray assays wherelabeled analytes become bound to discrete sites on the microarray.

Homogeneous probe dependent assays are also well known in the art andmay take advantage of the present invention. Examples of such methodsare energy transfer between adjacent probes (U.S. Pat. No. 4,868,103),the Taqman exonuclease assay (U.S. Pat. Nos. 5,538,848 and 5,210,015),Molecular Beacons (U.S. Pat. Nos. 5,118,801 and 5,925,517) and variousreal time assays (US Patent Application Publication 2005/0137388), allof which are incorporated by reference.

Antibodies labeled with dyes of the present invention may be used invarious formats. For example, an antibody with one of the dyes of thepresent invention may be used in an immunofluorescent plate assay or insitu analysis of the cellular location and quantity of various antigenictargets. Antibodies labeled with dyes may also be used free in solutionin cell counting or cell sorting methods that use a flow cytometer orfor in-vitro and in-vivo imaging of animal models.

The presence or absence of a signal may then be used to indicate thepresence or absence of the target itself. An example of this is a testwhere it is sufficient to know whether a particular pathogen is presentin a clinical specimen. On the other hand, quantitative assays may alsobe carried out where it is not so much the intention of evaluating if atarget is present but rather the particular amount of target that ispresent. An example of this is the previously cited microarray assaywhere the particular rise or fall in the amount of particular mRNAspecies may be of interest.

In another embodiment of the present invention, dyes that have beendisclosed above as well as dyes described previously in the literaturemay be attached to a carrier with a more general affinity. Dyes may beattached to intercalators that in themselves do not provide signalgeneration but by virtue of their binding may bring a dye in proximityto a nucleic acid. A further example is attachment of dyes to SDSmolecules thereby allowing dyes to be brought into proximity toproteins. Thus this embodiment describes the adaptation of a dye or dyesthat lack affinity to a general class of molecules may be adapted bylinking them to non-dye molecules or macromolecules that can convey suchproperties.

Various applications may enjoy the benefits of binding the dyes of thepresent invention to appropriate targets. As described above, stainingof macromolecules in a gel is a methodology that has a long history ofuse. More recent applications that also may find use are real timedetection of amplification (U.S. Pat. Nos. 5,994,056, 6,174,670 and USPatent Application Publication 2005/0137388, all of which are herebyincorporated by reference), and binding of nucleic acids to microarrays.In situ assays may also find use where the binding of dyes of thepresent invention is used to identify the location or quantity ofappropriate targets.

In other aspects, this invention provides a composition comprising asolid support to which is attached any of the above-described reactivecompounds. In some embodiments, the solid support comprises glassparticle, glass surface, natural polymers, synthetic polymers, plasticparticle, plastic surface, silicaceous particle, silicaceous surface,glass, plastic or latex beads, controlled pore glass, metal particle,metal oxide particle, microplate or microarray, or any combinationthereof. The aforementioned reactive group for attachment comprises ormay have comprised an electrophilic reactive group comprisingisocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine,4,6,-dichloro-1,3,5-triazines, mono- or di-halogen substituted pyridine,mono- or di-halogen substituted diazine, maleimide, haloacetamide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol,azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde groups, anucleophilic reactive group comprising reactive thiol, amine orhydroxyl, a nickel coordinate group, a platinum coordinate group, aterminal alkene or a terminal alkyne, and any combination of theforegoing. As in the case of other embodiments previously describedabove, a linker arm can be usefully positioned between the compound andthe reactive group, or between the solid support and the reactive group.

Reagent Kits

Commercial kits are valuable because they eliminate the need forindividual laboratories to optimize procedures, saving both time andresources. They also allow better cross-comparison of results generatedfrom different laboratories. The present invention thus additionallyprovides reagent kits, i.e., reagent combinations or means, comprisingall of the essential elements required to conduct a desired assaymethod. The reagent system is presented in a commercially packaged form,as a composition or admixture where the compatibility of the reagentswill allow, in a test kit, i.e., a packaged combination of one or morecontainers, devices or the like holding the necessary reagents, andusually written instructions for the performance of the assays. Reagentsystems of the present invention include all configurations andcompositions for performing the various labeling and staining formatsdescribed herein.

The reagent system will generally comprise (1) one or more dye of thepresent invention preferably in the form of concentrated stock solutionsin an aprotic dipolar solvent, for example, DMSO designed to targetspecific protein aggregate structures; (2) a buffer, such as Tris-HCl orphosphate buffer; (3) a positive control comprising both proteinaggregates and protein monomers in the state of solution or lyophilizedpowder; and (4) instructions for usage of the included reagents. Genericinstruction, as well as specific instructions for the use of thereagents on particular instruments, such as a wide-field microscope,confocal microscope, flow cytometer, high content screening instrument,microplate-based detection platform, RT-PCR instrument or standardfluorometer may be provided. Recommendations regarding filter setsand/or illumination sources for optimal performance of the reagents fora particular application may be provided.

The dyes, compounds and compositions of the present invention arefluorescently detectable or localized. Techniques and fluorescencemethods are well known in the art. A compilation of such techniques andmethods are set forth below in Table 3 which was obtained from Hawe etal., 2008.

TABLE 3 Fluorescence methods and their application with extrinsicfluorescent dyes for protein characterization. Application withNoncovalent Method Information Extrinsic Dyes Steady-state Spectralinformation Detection of protein fluorescence (emission spectrum andstructural changes by fluorescence intensity dye-protein interactionsTime-resolved Fluorescence lifetime Detection of protein fluorescencestructural changes by dye-protein interactions Anisotrophy Rotationalmotions Study of rotational (steady-state and dynamics Determination oftime-resolved size of dye-protein complexes Fluorescence TranslationalDetermination of size of correlation motions/diffusion dye-proteincomplexes spectroscopy (FCS) Fluorescence Visualization of Detection oflarge microscopy particles dye-protein complexes Determination of sizeand morphology of large aggregates, fibrils, etc.For an expert review on such fluorescence methods, see the entire abovecited publication by Hawe et al., 2008, pp. 1487-1499, the contents ofwhich are incorporated herein by reference.

Protein Aggregation Detection and Analysis

Fluorescence microscopy allows an early detection of changes in proteinsolutions, while minimizing alterations to the observed sample afterstaining with appropriate dyes. In protein formulations, the ability todetect protein aggregates at early time points with the dyes of thepresent invention can accelerate stability testing and reduce number ofsamples in long term stability studies. Fluorescence microscopy providesthe possibility of studying subtle changes in the aggregation state ofthe proteins, which is also of interest in medicine and biology,whenever protein characterization is needed. Also, fluorescencemicroscopy allows the characterization of high-concentration proteinformulations without dilution and with minimal impact on the protein'slocal environment. Furthermore, high-content screeningfluorescence-based imaging methods allow quantification of populationsof protein aggregates including number of branches, mean fiber length,mean fiber width, size distribution, polydispersity, kinetics offormation and kinetics of disassembly.

The present invention includes an example of IgG aggregate detectionusing dyes of the invention by fluorescence microscopy (Examples 2 and10; FIG. 1). The aggregate formation is barely visible before staining,but clearly becomes visible after staining.

The dyes of the invention are also capable of detecting a broader rangeof protein aggregates than the conventional amyloid detecting dyes, suchas thioflavin T (Thio-T) or congo red. These styryl dyes are able tosensitively detect protein aggregates, ranging in size (nanometers tovisually observable turbid solution to precipitates) and physicochemicalcharacteristics (e.g., soluble or insoluble, covalent or non-covalent,reversible or irreversible). Structurally altered proteins have a strongtendency to aggregate, often leading to their precipitation.Irreversible aggregation is a major concern for long-term storagestability of therapeutic proteins and for their shipping and handling.

The styryl dyes of the present invention are also able to detectaggregates at different stages of formation induced by various stresses,such as elevated temperature, agitation and exposure to extremes of pH,ionic strength, or various interfaces (e.g., air-liquid interface) andhigh protein concentration (as in the case of some monoclonal antibodyformulations), chemicals and protein-protein interactions (i.e.,PDI-insulin interaction). These fluorescent probes are able to detectbroad types and concentration ranges of proteins, in the presence ofexcipients, at different pH values (2˜10) and in the presence of saltsand buffers, exhibiting desirable detection limits and dynamic range,excellent sensitivity as well as linear response. This is exemplified bythe broad categories of proteins/peptides system in the presentinvention, including lysozyme, insulin, and IgG molecules, as well asserum proteins, such as β-lactoglobulin (BLG) and BSA. Therefore, thesenovel dyes are capable of providing quantitative analysis of proteinaggregates in a robust, high throughput fashion.

Thus, the present invention provides a method for detecting the presenceof aggregates of a protein in a sample. The method comprises

(a) combining the sample with any of the above-described compounds ormultidye compositions to form a dye-sample mixture;

(b) measuring the amount of fluorescence in the dye-sample mixture;

(c) comparing the amount of fluorescence determined in (b) with theamount of fluorescence in

-   -   (i) a mixture of the compound or multidye composition with a        control sample without aggregated protein, or    -   (ii) a mixture of the compound or multidye composition with a        known standard quantity of aggregated protein; and

(d) determining the aggregation of the protein in the sample based onthe comparison in (c).

In these methods, the standard quantity of aggregated protein recited in(c)(ii) can be prepared by any means known in the art. Examples includethe provision of a previously determined quantity of aggregated protein,or the preparation of a standard curve derived from measurements ofprotein aggregates and protein monomers in selected proportions. When astandard curve is utilized, the protein for the standard curve can bethe same or different protein as the protein in the sample.

The sample for this method is not limited to any particular composition.The sample can be from any prokaryote, archaea, or eukaryote, or from anenvironmental sample. In some embodiments, the sample is from a mammal,for example a bodily fluid of the mammal (e.g., blood [e.g., serum,plasma], bile, sputum, urine, or perspiration).

In other embodiments, the sample comprises a cell from the mammal. Insome of these embodiments, the cell is intact. Such an intact cell,either fixed (see, e.g., Example 28) or living (e.g., Example 29), canbe combined with the compound or multidye composition and thefluorescence is measured histologically. Here, the fluorescence can bemeasured by visual observation or by quantifying the amount offluorescent light emitted from the cell, by known methods.

Example 29 exemplifies embodiments utilizing living cells where acompound can be tested for an effect on the aggregation of proteins. Inthese embodiments, the cell is treated with a protein, for exampleamyloid beta peptide (e.g., amyloid beta peptide 1-42), known toaggregate in the cell. Such cells treated with the compound can becompared with cells not treated with the compound (the controlcomposition of (c)(ii) in the above-described methods) to determine theeffect of the compound on the aggregation of the protein in the cell.

The sample of these methods can also comprise homogenized cells from amammal that is part of a tissue from a mammal. In some embodiments, themammal has a disorder characterized by altered protein aggregation,e.g., Alzheimer's disease, Huntington's disease, Parkinson's disease,senile systemic amyloidosis, or a spongiform encephalopathy.

These methods can be utilized to detect any known form of aggregatedprotein, including but not limited to aggresomes, aggresome-likestructures, inclusion bodies, Lewy bodies, Mallory bodies,neurofibriliary tangles, or any combination thereof.

Protein Aggregation Kinetic Studies

Protein aggregation is an important phenomenon that alternatively ispart of the normal functioning of nature or has negative consequencesvia its hypothesized central role in neurodegenerative diseases. A keyin controlling protein aggregation is to understand the mechanism(s) ofprotein aggregation. Kinetic studies, including data curve-fitting, andanalysis are, in turn, keys to performing rigorous mechanistic studies.

The many approaches in the literature striving to determine the kineticsand mechanism of protein aggregation can be broadly divided into threecategories: (i) kinetic and thermodynamic, (ii) empirical, and (iii)other approaches. The large literature of protein aggregation can bedistilled down to five classes of postulated mechanisms: i) thesubsequent monomer addition mechanism, ii) the reversible associationmechanism, iii) prion aggregation mechanisms, iv) an “Ockham'srazor”/minimalistic model, and v) quantitative structure activityrelationship (QSAR) models (Morris et al., 2009). Correspondingequations derived from aggregation kinetic data can enlighten whichproposed mechanism is applicable to the specific protein.

Detection of aggregates at their nascent stages, such as intermediatesconsisting of a couple of monomers, is key in determining criticalnucleus size and aggregate growth mechanism. In addition, kineticstudies are also very helpful in screening excipients or inhibitors thatcan stop or suppress protein aggregation and in assessing enzymeactivity in various clinical and research settings. Hence, a sensitivekinetic assay in a robust, high-throughput manner is highly desirable inmechanism determination studies and in drug discovery. Most of thecurrent aggregate analysis technologies, unfortunately, are neithersensitive nor accurate enough to quantify nascent aggregates. Variousfactors affecting aggregation can be studied by these means; a number ofthese are described by Bondos and Bicknell (2003) and in addition, Table4 below is reproduced from this article (Table 1 therein) showingcomponents (including recommended concentrations) that might be used fordecreasing aggregation:

TABLE 4 Agents that may promote protein solubility Recommendedconcentration Additive range Kosmotropes MgSO₄ 0-0.4M (NH₄)₂SO₄ 0-0.3MNa₂SO₄ 0-0.2M Ca₂SO₄ 0-0.2M Weak kosmotropes NaCl 0-1M KCl 0-1MChaotropes CaCl₂ 0-0.2M MgCl₂ 0-0.2M LiCl 0-0.8M RbCl 0-0.8M NaSCN0-0.2M NaI 0-0.4M NaClO₄ 0-0.4M NaBr 0-0.4M Urea 0-1.5M Amino acidsGlycine 0.5-2% L-arginine 0-5M Sugars and Sucrose 0-1M polyhydricGlucose 0-2M alcohols Lactose 0.1-0.5M Ethylene glycol 0-60% v/v Xylitol0-30% w/v Mannitol 0-15% w/v Inositol 0-10% w/v Sorbitol 0-40% w/vGlycerol 5-40% v/v Detergents Tween 80 0-0.2% w/v Tween 20 0-120 μMNonidet P-40 0-1%

The method described above can be adapted to measure the kinetics ofprotein aggregation, e.g., by measuring fluorescence of the protein-dyemixture at various time points while aggregation is occurring. Thus, insome embodiments, the amount of fluorescence of the above-describedmethod is measured at preselected time intervals to detect formation ofprotein aggregates, wherein increasing fluorescence over time indicatesformation of protein aggregates.

These embodiments encompass two methods of applying the above-describeddyes into a kinetics study of protein aggregation, such as lysozyme andIgG aggregation, induced by various types of stress, including pH,shaking and temperature shift and in the presence or absence ofexcipient(s). The first method comprises the following steps: (1) applya stress to a protein formulation for a certain period of time; (2)release stress by switching off the stress, such as heat or harsh pH tofreeze or trap the aggregate formation; (3) fluorescence reading ofthese formulations by addition of selected dyes of the invention; (4)plot the relative fluorescence unit (RFU) vs. time curve and furtherprocess the kinetic curve to extract more desired information. Thismethod is beneficial for some proteins whose aggregation can besignificantly interfered with by probing dye binding (especially fornascent or intermediate aggregates, characterized by a much smallersurface area than those more matured aggregates) at stressed condition,which is minimized after the release of the stress.

The second method is more convenient compared to the first method.First, mix the dye with the protein formulation prior to the applicationof the stress; second, apply the stress and start recording thefluorescence response at various points of time; finally, plot arelative fluorescence unit (RFU) vs. time curve and possibly performfurther processing of the curve to extract more desired information.This method, though labor saving, much more robust and accurate in time,may not be applicable for some proteins if the dye blocks, promotes orinterferes with the addition of monomers to the aggregate intermediatesor polymerization of aggregate intermediates. However, notwithstandingthe mentioned caveats, the second method is generally preferred, sinceit allows for a simpler high throughput assay.

The measurement of fluorescence in these methods can be conducted usingany appropriate time interval between measurements, determined by adetermination of the time expected for the aggregation to occur in theparticular system being investigated. In some embodiments, thepreselected time intervals are less than 2 minutes. In otherembodiments, the preselected time intervals are less than 10 minutes. Instill other embodiments, the preselected time intervals are less than 1hour. In additional embodiments, the preselected time intervals are morethan 1 hour.

Methods of Evaluating Protein Formulation Stability Using AcceleratedStability Testing

Embodiments of the present invention are directed to reliable, time andcost-efficient methods for evaluating the relative chemical and physicalstability of a particular protein formulation. Thus, embodiments of theinvention are useful analytical tools for developing new proteinformulations with increased stability, as well as for use in evaluatingthe stability of newly prepared batches of known protein formulations inquality control procedures, or the like.

Embodiments of the present invention encompass a fully automated assayof protein stability that generally requires less than one week forevaluating protein formulations. The present invention method comprisespreparing two series of formulations, one formed before stress test(pre-stress formulations), another formed after stress test (post-stressformulations), followed by an adding aggregate detection reagent thatinclude one or more dyes of the present invention. The dye or dyes ofthe present invention may be used alone for this purpose oror they maybe used in conjunction with other commercial dyes, such as Nile red,thioflavin-T, ANS or Congo red. This is followed by comparing thefluorescence response of different formulations to rank the amount ofaggregates existing within individual formulations.

In one exemplification of this method, the following 6 steps may becarried out:

Step (1). A selected group of components, including, but not limited toexcipients, salts, buffers, co-solvents, metal ions, preservatives,surfactants, and ligands are collected and their stock solutions areprepared.

Step (2). Preliminary formulations comprising one or more componentsfollowing a standard design of experiment procedure aimed at generatingrelevant information are designed and the protein formulations,preferably containing the same concentration of protein are prepared.

Step (3). A stress such as heat, agitation, rotation, harsh pH,ultrasound, shearing or the like, is simultaneously applied externallyto multiple protein formulations under evaluation, which are held inindividual containers, preferably in separate wells of one microplate(s), which is preferably sealed, each with zero, one or more componentsof interests; meanwhile, the formulation with zero component ofinterests, but the same protein concentration as the formulations withcomponent (s) of interests can be prepared in a separately sealedcontainer in bulk quantity.

Step (4). After stress is released, the bulk protein formulation thathas zero components of interests is split and mixed with one or morecomponents of interests to make up similar formulations as thosesubjected to the stress test, preferably in wells of another microplate.Note that the later added components of interest solutions dilute theresulted non-stressed formulations, making them less concentrated astheir stressed counterpart; this can be adjusted later in the step wherethe probing dyes are added. These control formulations which have notexperienced the stress test allow accurate evaluation of the functionsof the components of interests during the stress test since componentsof interests themselves can affect the fluorescence response of proteinaggregates to some extent.

Step (5). A solution of the dye or dyes of the present invention (andthe buffer in which the dyes are dissolved) are added into the proteinformulations such that post-stress formulations are more concentratedthan that added to the stressed formulations to result in the sameconcentration of protein, components of interests and dye(s) for bothpre-stress formulation and post-stress counterpart. After an incubationperiod, the microplates are read in a conventional plate reader by, forexample, fluorescence intensity or fluorescence polarizationmeasurement.

Step (6). The formulations can be first evaluated within the group (i.e.either pre-stress or post-stress formulations), which are preferablytested in one microplate, by comparing formulations containing one ormore components of interests with that containing no components ofinterests. This method can eliminate the errors produced during thepreparation of different plates (the sample formulation plate(s) and thecontrol formulation plate(s), which can take 10˜60 minutes. Thenfluorescence ratio of each stress tested formulation to itscorresponding control without stress application can be furthercalculated. The function of components of interests during stress isevaluated by using the fluorescence ratio of components of interestsadded before application of stress vs. after application of stress usingzero components of interests as a reference. Therefore, the presentinvention is further directed to a method to evaluate components ofinterests that can stabilize or destabilize protein in order to optimizeprotein formulations.

The properties of the dyes of the invention allow their wide applicationin the protein/peptide formulation field, especially on ahigh-throughput technology platform. Compared with other fluorescentprobes, such as intrinsic tyrosine or externally added probes, such as1-anilino-naphthalene-8-sulfonate (ANS), Nile red or thioflavin-T, thedyes of the present invention are better capable of providingquantitative analysis of protein aggregates in a robust, high throughputfashion and are applicable to more categories of proteins under variousconditions. In some instances two or more dyes of the present inventionare applied to a sample. This facilitates detection of the broadestrange of protein aggregates since these means provide that if one dyedoes not bind a particular aggregate, another can compensate for thisdeficiency.

Protein Stability Shift Assay Based on Fluorescent Detection of ProteinAggregation Using Exogenously Added Fluorophores

Protein stability can be altered by various components discussed inprotein formulation embodiments, including, but not limited toexcipients, salts, buffers, co-solvents, metal ions, preservatives,surfactants, and ligands. Protein stability can be shifted by variousstresses, including elevated temperature, which is often referred to asa thermal shift or by addition of chemical denaturants, such as urea,guanidinium isocyanate or the like. A protein stability shift assay hasa wide spectrum of applications in, but not limited to investigation ofprotein refolding conditions, optimization of recombinant proteinexpression/purification conditions, protein crystallization conditions,selection of ligand/drug/vaccine/diagnostic reagents and proteinformulations.

The classic thermal shift technologies based on protein aggregatedetection utilize a melting point device to raise the temperaturestepwise, coupled with aggregation detection technologies, such as lightscattering technology (an example includes but is not limited todifferential static light scattering (DSLS)) to monitor proteinaggregation. This type of technology usually requires a high proteinconcentration, therefore, it is not cost effective. In addition, itcannot work effectively on formulations with low protein concentrationsor finalize protein formulations which require a very low detectionlimit for aggregates (typically ˜1-5%), which is usually beyond thedetection limit of these classic technologies.

Thermofluor® (J&J, 3-Dimensional Pharmaceuticals, Inc, Exton, Pa., U.S.Pat. No. 6,020,141 [“the '141 patent”]) is a biophysical technique usedto study (relative) protein stabilities. The solution of protein isheated up stepwise from room temperature to ˜95° C. and the fluorescenceis monitored at each step. The rising temperature causes proteinunfolding and the fluorophore (SYPRO Orange® [Invitrogen] or ANS)partitions itself into the melted protein and hence the overall effectis an increase in fluorescence with increasing temperature. If a drug orligand is included which binds to the protein, the mid-point of thecurve can shift, arising from stabilizing or destabilizing effects(e.g., ligand binding). Thermofluor® can rank binding affinity in arapid, HTS manner and help setup structure-activity relationship.However, this particular methodology is related to both denaturation ofproteins as well as subsequent aggregations of the denatured proteinsand the patent clearly indicates that the focus is on the unfolding anddenaturation of proteins and as described in column 16, lines 25-56, thefluorescent probes chosen for application of this method are drawn fromcompounds that are “capable of binding to an unfolded or denaturedreceptor”. However, some of the compounds that are listed (ANS, bis-ANSand JCVJ) are known to bind to aggregates (Lindgren et al., 2005) and assuch no particular emphasis is laid upon distinguishing betweendenaturation and aggregation events. In contrast, the present inventionis specifically directed towards aggregation detection.

As such, one of the embodiments of the present invention encompasses anovel thermal shift assay in which protein is heated up stepwise fromroom temperature to ˜95° C. using a device, including, but not limitedto, a microplate reader, a thermocycler, a melting device or similarequipment, preferably with a heating stage that can raise temperaturestepwise and record fluorescence change simultaneously, and thefluorescence of externally added dyes of the present invention ismonitored at each heating step. Since the dyes that are used in thepresent invention selectively interact with protein aggregates and nothydrophobic domains exposed by protein unfolding, the increase influorescence with increasing temperature is not due to protein unfoldingas seen in the technique described in the '141 patent, but rather is dueto protein aggregation. Therefore, this particular embodiment of thepresent invention can be applied to directly targeting at rankingcomponents, including, but not limited to, excipients, salts, buffers,co-solvents, metal ions, preservatives, surfactants, and ligands inprotein stabilization by preventing protein aggregation to improveformulations, or to screening drugs (inhibitors) preventing proteinaggregates found in some diseases, including, but not limited to,organic synthetic compounds, peptides and proteins (recombinant ornatural source). For most proteins, unfolding directly precedes theiraggregation. Hence, similar to the unfolding-based Thermofluor®technique, the aggregation-based thermal shift assay technology embodiedin this present invention also has the potential to being applied toranking the effect of additives on protein stability. Its applicationcan thus be expanded to more broad fields, including, but not limitedto, investigation protein refolding conditions, optimization ofrecombinant protein expression/purification conditions, proteincrystallization conditions, and selection of ligands, drug, vaccine anddiagnostic reagents.

Thus, fluorescence can be measured at one or more different temperaturesafter forming the first mixture and the second mixture. Such differenttemperatures can be selected from temperatures ranging from about 4° C.to about 100° C. Further, fluorescence measurements can be carried outas a series of discrete temperatures, where the measuring steps arecarried out after incubation at each of the different discretetemperatures, or while changing temperatures.

Another useful method of the present invention is a method fordetermining whether a test compound decreases aggregation of a protein.The above-described method can be utilized, where a test compound isadded to a portion of the dye-sample mixture of (a) and the fluorescenceof the portion with the test compound is compared to the fluorescence ofthe portion without the test compound to determine whether the testcompound decreases aggregation of the protein, wherein decreasedfluorescence in the portion with the test compound indicates that thetest compound decreases aggregation of the protein.

The test compound is not limited to any particular class of compound.Nonlimiting examples include a kosmotrope, a chaotrope, an amino acid, apeptide, a reducing agent, a carbohydrate, a detergent, a surfactant, azwitterion or a polyhydric alcohol, or any combination thereof. Any ofthese test compounds can have a range of concentrations from about 0molar to about 2 molar, a range of pH values from about 4 to about 10.The test compound can also comprise a storage buffer for said protein.Such storage buffer can comprise a set of buffer formulations with arange of concentrations from about 0 molar to about 2 molar, a range ofpH values from about 4 to about 10, and any combinations thereof.

Chaperone/Anti-Chaperone Activity Assays

Chaperone and anti-chaperone function oppositely in the sense that onehelps prevent aggregates and the other helps induce aggregate formation.To assay activity of the opposite functions, one needs to quantitativelyanalyze the substrate aggregate change with time. The present inventionuses methods described above in the PDI/thioredoxin activity assay toanalyze chaperone/anti-chaperone activity, which has similar advantagesover methods based on other aggregate detection technologies,particularly turbidity and back-scatter methods. The present inventionalso encompasses a kit or kits comprising similar components as the PDIisomerase activity kit (s) included in the present invention. Assays canbe devised to monitor assembly or disassembly of protein aggregates orboth.

Thus, in some embodiments of the above-described method,

(A) the protein is a substrate for a chaperone;

(B) the dye-sample mixture of step (a) is subjected to a stress for atime and under conditions sufficient to induce aggregation of theprotein; and

(C) the amount of fluorescence determined in (b) is compared to theamount of fluorescence from the protein with the compound or multidyecomposition subjected to the same stress without the sample. In theseembodiments, a decrease in fluorescence of the stressed dye-samplemixture with the sample when compared to the fluorescence from theprotein with the compound or multidye composition but without the sampleindicates that the sample comprises the chaperone.

This method can utilize any chaperone now known or later discovered,including chaperones that are small heat-shock proteins (sHSPs), as theyare known in the art. Examples of chaperones include HSP33, HSP60,HSP70, HSP90 or HSP100, GRP94, GRP170, calnexin, calreticulin, HSP 40,HSP47 and ERp29, GroEL, GroES, HSP60, Cpn10, DnaK, DnaJ, Hsp70, Hsp71,Hsp72, Grp78 (BiP), PDI, Erp72, Hsx70, Hdj1, Hdj2, Mortalin, Hsc70,Hsp70-A1, fHtpG, C62.5, Hsp90 alpha, Hsp90 beta, Grp94, ClpB, ClpA,ClpX, Hsp100, Hsp104, Hsp110, TRiC, alpha crystallin, HspB1, Hsp 25,Hsp27, clusterin, GrpE, Trigger Factor, and Survival of Motor Neuron(SMN1, SMN2), or any combination thereof. The substrate can comprise anychaperone substrate now known or later discovered, including but notlimited to β-lactoglobulin, citrate synthase, lysozyme, immunoglobulin,CRYBB2, HSPB8, CRYAA, TGFB1I1, HNRPD or CRYAB, or any combinationthereof. The reaction mixture can be incubated for a period of time fromabout 15 to about 60 minutes. The stress can be an elevated temperature,preferably, from about 37° C. to about 95° C. Alternatively, the stresscan be a chaotropic agent, such as guanidine-HCl or urea, or both. Theconcentration of the chaotropic agent can be from about 4 to 8 M.Moreover, a plurality of these methods can be performed in parallel.

Analogously, the invention methods can be utilized to identifyanti-chaperone activity. Here, the methods described above are utilized,where

(A) the protein is a substrate for an anti-chaperone; and

(B) the amount of fluorescence determined in (b) is compared to theamount of fluorescence from the protein with the compound or multidyecomposition without the sample. In these methods, an increase influorescence of the dye-sample mixture when compared to the fluorescencefrom the protein with the compound or multidye composition but withoutthe sample indicates that the sample comprises the anti-chaperone.

High-Throughput Fluorometric Assay for Measuring Aggregates Formed byMembers of the Thioredoxin Superfamily

Thioredoxins and related proteins act as antioxidants by facilitatingthe reduction of other proteins by cysteine thiol-disulfide exchange.Such exchanges can lead to intermolecular bridges being formed, therebyforming covalently linked aggregates. Thioredoxins are characterized atthe level of their amino acid sequence by the presence of two vicinalcysteines in a CXXC motif. These two cysteines are the key to theability of thioredoxin to reduce other proteins. A number of differentfamilies (thioredoxins, protein disulfide isomerases [PDI's] andglutaredoxins) form what can be considered the thioredoxin superfamily.With regard to the glutaredoxins, they share many of the functions ofthioredoxins, but are reduced by glutathione rather than a specificreductase and may be assayed by the described methods of the presentinvention.

Thus, methods are disclosed to measure the activity of thioredoxin-likeenzymes by detecting the induction of aggregates formation by utilizingany of the dyes described above. The above-described method fordetecting a protein aggregate can be utilized, where

(A) the protein is a substrate for a member of the thioredoxinsuperfamily;

(B) a reducing agent is included in the dye-sample mixture of (a); and

(C) the dye-sample mixture of step (a) is incubated for a period of timesufficient to reduce disulfide bonds in the protein. In these methods,an increase in fluorescence of the dye-sample mixture when compared tothe fluorescence from the protein with the compound or multidyecomposition without the sample indicates that the sample comprises themember of the thioredoxin superfamily.

Substrates here include, but are not limited to, insulin,hypoxia-inducible factor, prolyl 4-hydroxylase, HIV gp120, TXNIP, ASK1,collagen type I, alpha 1 and glucocorticoid receptor. In someembodiments, insulin is used as a substrate at a concentration of lessthan 0.2 mM. This method can be used to measure the amount of activityin a sample, identify the suitability of proteins as substrates for suchactivity, and to screen for inhibitors of these enzymes. This method mayalso be used to test the ability of a particular protein to be used as asubstrate by a member of the thioredoxin superfamily to form aggregates.This method also allows an accurate assay of multiple samples, such assamples from patients, or therapeutic agents for drug discovery. Themethod can be used in a high throughput manner using a microplate, asreflected in the insulin aggregate detection example included in thepresent invention.

The reducing reagent concentration should be optimized to reduce thesubstrate disulfide bonds without minimizing the competing chemicalreaction. The reducing reagents may include, but are not limited toglutathione, dithiothreitol (DTT), dithioerythritol, β-mercaptoethanol,thioglycolate, and cysteine, with DTT being a preferred embodiment. Apreferred DTT concentration is less than 10 mM, and more preferably lessthan 1 mM. The assay buffer can include those buffers that stabilizethioredoxin superfamily members and their substrates, with optimized pH,salts, chelating agents (e.g. EDTA, and the like), dyes, and potentiallyorganic solvents such as DMSO.

When testing for the presence or amount of a particular member of thethioredoxin superfamily in a sample (or for overall activity), a varietyof sources may be used that include biological tissues, biologicalfluids and cells. Thus for instance, samples may include cellsup-regulating PDI during hypoxia or cells with surface expressed PDI,including endothelial cells, platelets, lymphocytes, hepatocytes,pancreatic cells and fibroblasts. The sample may also include athioredoxin superfamily member complexed with other proteins, such asPDI complexed with hypoxia-inducible transcription factor HIFa. Samplesmay also include fragments of a member of the thioredoxin superfamily aswell as recombinant forms of these members, and in vitro proteinsynthesis reactions that are presumed to have generated such proteins.

These methods may also find utility in identifying modulators ofthioredoxin superfamily activity; such modulators can comprise enzymemimetics, interacting proteins, competitive inhibitors, small molecularinhibitors, and the like.

The method may also comprise the use of appropriate controls for thesample, including controls that do not include any thioredoxinsuperfamily member activity as well as controls that do not include anyreducing reagents. These controls can be used as background to besubtracted from gross signal to gain net signal induced by the enzymeactivity.

A preferred addition sequence of the present invention is: (1) Substrateand related buffers; (2) Dye(s) dissolved in organic solvent(s), (3) PDIor similar thioredoxin-like enzyme (s) and related buffers; (4) Reducingreagent (s). The enzyme(s) and reducing reagents are preferred to beadded with a multi-channel addition device that can simultaneously addreducing agent into the multiple assay containers, such as wells of amicroplate to minimize the time interval between the addition of enzymeand the reducing reagent. This may be important for kinetic assays undersome circumstances since PDI and similar thioredoxin-like enzymes caninduce enzymatic reaction in the absence of reducing agent, especiallywith a high concentration of enzyme or substrate or both. This canminimize the background levels. The multi-channel addition device canminimize the background levels derived from the foregoing effects it mayalso minimize timing errors among the multiple samples to be analyzed,which can minimize statistical deviation among the samples.

In addition to the methods described above, the thioredoxin superfamilyaggregation assays can be formulated into kits comprising one or morethioredoxin superfamily members, appropriate substrates, buffers,reducing agents and one or more dyes of the described in FIG. 1 as wellas instructions for their use. These kits may be used for any of theapplications described above.

Such member of the thioredoxin superfamily (a) can comprise a proteindisulfide isomerase, a thioredoxin or a glutaredoxin, and combinationsthereof. The substrate (b) in this method can comprise insulinribonuclease, choriogonadotropin, coagulation factor, glucocorticoidreceptor or HIV gp 120, and combinations thereof. The reducing agent (c)can be selected from the group comprising dithiothreitol (DTT),Tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl) ordithioerythritol (DTE), and combinations thereof. The reaction mixturecan be preferably incubated for a period of time from about 15 to about60 minutes. The protein disulfide isomerase can comprise PDI, ERp57,PDIp, ERp72, P5, PDIr, ERp28/29, ERp44, ERjd5/JPDI or ERp18, andcombinations thereof.

This method can further comprise the step of terminating the reactionprior to the measuring step (iii) by adding hydrogen peroxide to theincubating reaction mixture. As in the case of earlier describedembodiments of this invention, a plurality of such methods can beperformed in parallel.

Assaying Various Enzymatic Activities and Post-TranslationalModifications by Monitoring Protein Aggregation Status

With respect to various pathological disorders, abnormal proteinaggregates are often sequestered into intracellular protein depositssuch as aggresomes, aggresome-like structures, inclusion bodies. Lewybodies or Mallory bodies (Stefani, 2004; Garcia-Mata et al., 2002).These may trigger in turn the expression of inflammatory mediators, suchas cyclooxygenase 2 (COX-2) (Li et al., 2004). Disruption of theubiquitin-proteasome pathway, as for example, thru impairment ofubiquitin hydrolase activity, triggered by modulators such as Δ12-PGJ2,lactacystin β-lactone or MG-132 can readily be analyzed directly incells using the disclosed methods to detect intracellular proteindeposits as well as in either cell-based or biochemical assays forscreening of other selective inhibitors of the ubiquitin-proteasomepathway that lead to protein aggregation.

The principle advantages of the delineated approach relative to use ofconventional substrates of ubiquitin hydrolase activity, such asubiquitin-7-amino-4-methylcoumarin (ubiquitin-AMC), include employmentof a natural protein substrate in the assay as well as an inherentsignal amplification, arising from the formation of polymerized amyloidfibrils as reporters. Examples of potential protein substrates useful inthis regard include, but are not limited to, immunoglobulin,α-synuclein, synphilin-1, TCRα, P23H mutant of rhodopsin, ΔF508 mutantof CFTR, amyloid-β, prion protein, Tau, SOD1, Ig light chains, ataxin-1,ataxin-3, ataxin-7, calcium channel, atrophin-1, androgen receptor,p62/sequestosomel (SQSTM1), Pael receptor, serum amyloid A,transthyretin, β2-microglobulin, apolipoprotein A-1, gelsolin, atrialnatriuretic factor, lysozyme, insulin, fibrinogen, crystallins,surfactant protein C, lactoferrin, βig-h3, PAPB2, corneodesmosin,neuroserpin, cochlin, RET, myelin, protein 22/0, SCAD, prolactin,lactadherin, p53, procalcitonin, cytokeratins, GFAP, ATP7B, prolylhydroxylase PHD3, presenilin, and huntingtin. Additionally, proteinsspecifically engineered to be unstable or highly prone toself-association into aggregates may be employed as substrates using thedisclosed assay methods.

With respect to coupled enzyme reactions the product of one reaction isused as the substrate of another, more easily-detectable reaction. Thecited compositions and methods are especially advantageous in thedevelopment of biochemical assays involving coupled reactions leading tothe formation of protein aggregates. In this instance no meaningfulphysiological relationship between the activity being monitored and thegeneration of the aggregated protein-dye reporter is explicitlyrequired. The protein aggregate-dye complex is simply serving as anindicator to establish the amount of product formed in a particularcatalytic reaction. For example, a protein substrate may be employedthat is marginally stable under the specified solution conditionsemployed in the assay. When this substrate is acted upon by a histoneacetyltransferase, a particular lysine residue becomes acetylated, theoverall protein structure is destabilized and the protein undergoes aconformational change resulting in its aggregation. The dyes describedin this disclosure are then able to bind to the aggregates, generating afluorescent signal. While illustrated with histone acetyltransferase, awide range of activities that could potentially modify a substrateprotein, leading to its structural destabilization under the assayconditions employed, could be performed by similar approaches. Inaddition activities that do not directly modify the substrate proteincan also be considered. For instance, an enzyme activity that leads tothe acidification of the assay buffer could in turn lead todestabilization of the substrate protein structure and its aggregation.

Separation of Protein Aggregates from Protein Monomers

Those skilled in the art will appreciate that the present invention isapplicable to the separation or isolation of protein aggregates fromother protein forms, notably protein monomers. The dyes described aboveare useful in subtraction of protein aggregates from protein monomers.

Thus, the present invention provides a method for separating aggregatesof a protein from monomeric forms of the protein. The method comprises

(a) combining the sample to the solid support of claim 25 underconditions where aggregates of the protein preferentially bind to thecompound;

(b) separating sample protein bound to the solid support from unboundprotein. In these methods, protein bound to the solid support issubstantially aggregates and unbound protein is substantially monomers.

In carrying out the above isolation method, the solid support cancomprise glass particle, glass surface, natural polymers, syntheticpolymers, plastic particle, plastic surface, silicaceous particle,silicaceous surface, glass, plastic or latex beads, controlled poreglass, metal particle, metal oxide particle, microplate or microarray,and combinations of any of the foregoing.

Preferred embodiments are described in the following examples. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example 1. Testing Compounds for Ability to Sense Protein Aggregation

Fluorescent readings were carried out in 50 mM Tris-HCl, pH 7.8 using 10μM dye. When present, 1 μM recombinant human α-synuclein (ASN,Sigma-Aldrich, St. Louis, Mo.) as monomers, or aggregated as describedin van Raaij et al. (2006) was included. Fluorescence excitation andemission spectra were collected on a Cary Eclipse fluorescencespectrophotometer (Varian, Australia). Fluorescence spectra weremeasured with excitation and emission slit widths set to 5 nm, and at aconstant PMT voltage. Spectroscopic measurements were performed instandard quartz cells. All measurements were made at the respectiveexcitation maxima of each dye. All measurements were carried out at roomtemperature. Results are summarized in Tables 1 and 2.

Example 2. Fluorescence Sensitivity of Different ProteinAggregate-Sensing Dyes in the Presence of Excipients

IgG aggregate was prepared by adjusting 5.83 mg/ml of purifiedgoat-anti-mouse IgG (H&L, Pel Freez, Rogers, Ark.) to pH 2.7 using HCland incubating at 22° C. for 24 hours. The assay was performed using 2.8μM IgG, either native or aggregated, and a dye concentration of 0.625μM. The protein and dye were mixed together for 15 minutes at 22° C.,then further incubated in the presence of the excipients shown in Table5. The fluorescence intensity of S-25, Tol3 and Y2150 were determinedwith a FLUOstar OPTIMA plate reader (BMG LABTECH) at excitationwavelength of 550 nm and emission wavelength of 610 nm; while thefluorescence intensity for thioflavin-T was determined using aSpectraMAX GeminiXS (Molecular device, with Softmax Pro 7.0) using anexcitation wavelength of 435 nm and emission wavelength of 495 nm. Thefluorescence enhancement (aggregate/native IgG) is shown.

TABLE 5 Effect of various concentrations of various excipients onfluorescence of four dyes in the presence of aggregated IgG. Excipients& Concentrations S25 TOL3 Y2150 Thio-T Sodium Chloride, 10 mM 14.0 16.014.4 1.6 Sodium Chloride, 100 mM 13.6 16.2 11.3 1.3 Sodium Chloride,1000 mM 11.7 17.4 15.0 2.7 Calcium Chloride, 10 mM 9.7 14.9 12.4 3.1Calcium Chloride, 50 mM 9.6 13.9 14.7 1.5 Calcium Chloride, 200 mM 6.714.8 13.9 1.7 Ammonium Sulfate, 10 mM 15.4 15.6 12.4 2.8 AmmoniumSulfate, 100 mM 14.6 13.4 12.5 2.6 Ammonium Sulfate, 300 mM 13.3 16.914.6 1.4 Sorbitol, 100 mM 16.4 20.0 17.3 3.0 Sorbitol, 300 mM 21.0 19.215.6 1.9 Sorbitol, 600 mM 25.4 29.3 18.7 3.6 Mannitol, 100 mM 16.7 17.511.2 3.1 Mannitol, 300 mM 15.2 25.2 13.8 3.7 Mannitol, 600 mM 20.9 27.517.7 1.8 Trehalose, 100 mM 17.8 18.9 14.0 2.2 Trehalose, 300 mM 32.120.1 19.4 0.2 Trehalose, 600 mM 30.1 30.4 18.9 4.8 Lactose, 100 mM 23.019.9 17.5 1.2 Lactose, 300 mM 38.9 34.6 31.0 1.4 Ascoric Acid, 1 mM 13.915.4 14.5 1.5 X100, 0.01% 19.2 6.2 4.1 5.3 X100, 0.2% 7.3 3.4 2.6 6.6X100, 1% 2.9 1.9 1.7 3.4 Arginine, 200 mM 14.8 18.6 14.4 1.4 Arginine,500 mM 13.5 17.6 14.3 2.0 Glycine, 0.5% 14.1 16.3 12.5 3.1 Glycine, 2%15.1 15.5 19.0 3.2 Tween 20, 0.01% 70.8 8.5 5.5 4.6 Tween 20, 0.2% 26.73.4 2.6 2.6 DTT, 1 mM 13.2 13.8 11.3 1.6 Average 19.3 17.6 14.8 2.9

Example 3. Synthesis of S25 (a) Preparation of 6-methylsulfonyloxyhexylmethylsulfonate (Compound 1)

A solution of 1,6-hexanediol (13.15 g, 111.3 mmol) in 70 mL of anhydrouspyridine was cooled to 0° C. using ice bath. To this methanesulfonylchloride (27 g, 235.7 mmol) was slowly added under mixing such that thetemperature was maintained at 5-6° C. The combined mixture was stirredovernight at the temperature below 10° C. and the precipitate formed wasfiltered off, washed with 20% HCl (3×), water (3×), 5% solution ofsodium bicarbonate (3×), and then again with water (3×). Product wasdried under vacuum to obtain Compound 1 as a white solid (yield 32.8%).The structure of Compound 1 is given below:

(b) Preparation of Compound 2

A mixture of 4-methylpyridine (3.06 g, 32.9 mmol) and Compound 1 (4.11g, 15 mmol) was heated at 120° C. for 3 hours. The reaction mixture wascooled and then 4 mL of isopropyl alcohol was added and the combinedmixture was refluxed for an hour. After cooling the precipitate wascollected by centrifugation, washed with isopropyl alcohol (40 mL, 3×),followed by diethyl ether (40 ml, 3×) and dried under vacuum overnightto provide Compound 2 (yield 85%) which was then used without anyfurther purification. The structure of Compound 2 is given below:

(c) Preparation of S-25

To a suspension of Compound 2 (1.38 g) in n-butanol (15 mL),p-dimethylaminobenzaldehyde (0.9 g) was added and the combined mixturewas stirred until it became homogeneous. To this mixture ˜24 drops ofpiperidine was added and it was refluxed for 4.5 hours. Upon cooling,the precipitate formed was collected by centrifugation, washed withisopropyl alcohol (40 ml, 3×), diethyl ether (40 ml, 2×) and dried undervacuum to provide dye S25 in a yield of about 68%. Abs=485 nm, Em=613nm. The structure of S25 is given below:

Example 4. Synthesis of Tol3 (a) Preparation of Compound 3

A mixture of 3,4-dimethylpyridine (1.18 g, 11 mmol) and1,10-diiododecane (1.97 g, 5 mmol) was alloyed during 3 hours at 120° C.To the reaction mixture 5 mL of isopropyl alcohol was added and themixture was refluxed for an hour. Upon cooling, the solvent wasdecanted, and the residue thus obtained was washed with cold diethylether (40 ml, 2×), followed by centrifugation to remove residualsolvents. The solid obtained was then dissolved in methanol (˜4 mL) anddropwise added to cold diethyl ether. Precipitated product was collectedby centrifugation, washed with diethyl ether (40 ml, 3×) and dried undervacuum to provide Compound 3 in 88% yield. This product was used withoutany further purification. The structure of Compound 3 is given below:

(b) Preparation of Tol3

A mixture of Compound 3 (0.61 g), p-dimethylaminobenzaldehyde (0.3 g)and 6˜8 drops of piperidine in 5 mL of n-butanol was refluxed for 4hours. After cooling the precipitated solid was collected bycentrifugation, washed first with isopropyl alcohol (40 ml, 3×), diethylether (40 ml, 2×) and then again isopropyl alcohol (40 ml, 1×) anddiethyl ether (40 ml, 3×). The product was dried under vacuum to providedye Tol3 in 82% yield. Abs=471 nm, Em=611 nm. The structure of Tol3 isgiven below:

Example 5. Synthesis of S43 (a) Preparation of1,1′-(1,2-phenylenebis(methylene))bis(4-methyl pyridinium) bromide(Compound 4)

A mixture of 4-methylpyridine (1.02 g) and 1,2-bis-bromomethyl-benzene(1.32 g) was heated during 2.5 hours at 120° C. To the reaction mixture5 mL of isopropyl alcohol was added and the mixture was refluxed for 2hours. After cooling the product was filtered, washed with diethyl etherand dried under vacuum to provide Compound 4 in 87% yield. The structureof Compound 4 is given below:

(b) Preparation of S43

A mixture of Compound 4 (0.45 g), p-dimethylaminobenzaldehyde (0.3 g)and 6 drops of piperidine in 5 mL of n-butanol were refluxed for 4hours. After cooling the product was filtered and washed with isopropylalcohol and diethyl ether. The residue obtained was recrystallized fromthe DMF-methanol mixture to provide S43 in 72% yield. Abs=527 nm, Em=637nm. The structure of S43 is given below:

Example 6. Synthesis of Yat 2134 (a) Preparation of1,1′-(butane-1,4-diyl)bis(4-methylpyridinium) iodide (Compound 5)

A mixture of 4-methylpyridine (1.02 g) and 1,4-diiodobutane (1.55 g) in5 mL of dioxane was refluxed for 8 hours. The obtained salt wasprecipitated with diethyl ether and filtered. The precipitate was washedwith ether and dried under vacuum to provide Compound 5 in 91% yield.This product was used without any further purification. The structure ofCompound 5 is given below:

(b) Preparation of Yat 2134

This procedure was carried out as described previously in step (b) ofExample 3 with Compound 5 (0.5 g), piperidine (˜6 drops), p-diethylaminobenzaldehyde (0.36 g) and n-butanol (5 mL). Purification was carried outby recrystallization from DMF-methanol mixture to provide Yat 2134 in70% yield. Abs=500 nm, Em=620 nm. The structure of Yat 2134 is givenbelow:

Example 7. Synthesis of Yat 2148

A mixture of Compound 4 [0.45 g, obtained in step (a) of Example 3],p-diethylaminobenzaldehyde (0.36 g) and 6 drops of piperidine in 5 mL ofn-butanol was refluxed for 4 hours. Upon cooling the product wasfiltered and washed with isopropyl alcohol and diethyl ether. The crudedye obtained was recrystallized from the DMF-methanol mixture to provideYat 2148 in 69% yield. Abs=520 nm, Em=632 nm. The structure of Yat 2148is given below:

Example 8. Synthesis of Yat 2149 (a) Preparation of1,1′-(2,2′-oxybis(ethane-2,1-diyl))bis(4-methylpyridinium) chloride(Compound 6)

A mixture of 4-methylpyridine (1.02 g) and 0.72 g of1-Chloro-2-(2-chloro-ethoxy)-ethane (0.72 g) was heated at 120-130° C.for 3-4 hours. To the reaction mixture 5 mL of isopropyl alcohol wasadded and the mixture was refluxed for an hour. Upon cooling the productwas filtered and washed with diethyl ether to provide Compound 6 in 81%yield. This product was used without any further purification. Thestructure of Compound 6 is given below:

(b) Preparation of Yat 2149

This procedure was carried out as described previously in step (b) ofExample 3 with Compound 6 (0.33 g), piperidine (˜6 drops),p-diethylamino benzaldehyde (0.36 g) and n-butanol (5 mL). After coolingthe dye was precipitated with isopropyl alcohol or diethyl ether. Inorder to obtain the iodide salt, a saturated aqueous solution of KI(0.34 g) was added to the dye solution in methanol. After cooling, theproduct was filtered, washed with isopropyl alcohol, diethyl ether anddried under vacuum to provide Yat 2149 in 65% yield. Abs=502 nm, Em=614nm. The structure of Yat 2149 is given below:

Example 9. Synthesis of Yat 2150

This procedure was carried out as described previously in step (b) ofExample 2 with Compound 3 (0.61 g), piperidine (˜5 drops),p-diethylamino benzaldehyde (0.36 g) and n-butanol (5 mL). Purificationwas carried out by recrystallization from DMF-methanol mixture toprovide Yat 2150 in 71% yield. Abs=485 nm, Em=612 nm. The structure ofYat 2150 is given below:

Example 10 Monitoring Protein Stability in Two Different BufferFormulations

Goat anti-mouse IgG from Vector Labs (1.5 mg) was resuspended in 150 μldeionized water (dH₂O). Phosphate was removed from the IgG using anAmbion NucAway spin column, following the manufacturer's instructions,briefly the column was resuspended in 700 μl dH₂O and allowed to hydratefor 60 minutes. Excess liquid was removed by centrifugation at 700×g for2 minutes. The column was placed in a fresh collection tube and thesample was carefully loaded on the center of the column. The IgG waseluted by centrifugation at 700×g for 2 minutes. The purified IgG wasdiluted 10 fold in either 100 mM HCl or 12 mM phosphate pH 7.4, 150 mMsodium chloride. The samples were incubated for 18 hours at 37° C. Thesolutions were stained with a final concentration of 100 mM MES, pH 6,0.25 mg/ml IgG, 3 μM S-25 and 3 μM Tol3 (1:1 ratio) for at least 15minutes. The stained protein was spotted on the surface of a glassmicroscope slide and overlaid with a cover slip, sealed with nail polishand observed using a BX51 microscope (Olympus, Tokyo, Japan). Imageswere acquired with a 40× objective lens (Olympus). Fluorescent imageswere acquired using a Texas Red filter set (Chroma Technoloogy Corp.,Rockingham, Vt.). FIG. 1 shows that fibrils were formed in HCl solution,but not in the neutral phosphate buffer. The fibrils formed exhibitedfluorescence that was bright and specific to the fibers using the S-25and Tol3 dye mixture. There was little or no fluorescence when fibrilshad not formed.

Example 11. Binding Curve of Different Fluorescent Probes to 20 μM ofAggregated Lysozyme Protein

Lysozyme aggregates were formed by dissolving Lysozyme in 10 mM HCl tomake a 1 mM Lysozyme solution (14.8 mg/ml). The Lysozyme solution washeated to 65° C. with shaking at 750 rpm in an Eppendorf thermomixer for90 hours. The lysozyme was diluted to 20 μM in a 50 mM potassiumphosphate solution containing different concentrations of a mixture ofthe dyes S-25 and Tol3. The aggregate was incubated for 15 minutes priorto measuring the fluorescence using a BioTek SynergyMx plate scanner,with excitation set at 515 nm and emission set to 603 nm, both with a 9nm slit-width. Readings were taken in at least triplicate in a GreinerμClear black, clear bottom 96-well microplate. As can be seen in FIG. 2,there is little or no signal generated with up to 1 nM of each of thedyes. Above 1 nM, the signal increases until about 1 μM each of thedyes, at which point no further signal increase is observed. Thisindicates that above 1 μM S-25 and 1 μM Tol3, the fluorescence of 20 μMaggregated Lysozyme is dependent on aggregate concentration, and not dyeconcentration.

Example 12. pH Sensitivity of Fluorescence Response to AggregatedLysozyme

Chicken egg white lysozyme (Sigma-Aldrich) was dissolved at 1 mM in 10mM HCl. This monomer solution was stored at 4° C. Lysozyme aggregate wasformed by shaking the protein solution at 750 rpm in a Thermomixer(Eppendorf) at 65° C. for 90 hours. The aggregation process wasmonitored by thioflavin T binding and after saturation of thefluorescence signal (for lysozyme after 90 hrs), the aggregate solutionwas also stored at 4° C.

FIG. 3A shows the effect of pH on the aggregation-specific fluorescenceof the dyes S-25 and Tol3, as determined by incubation of 4 μMaggregated lysozyme, 4 μM monomer lysozyme (or 8 μM lysozyme monomeralone) with 0.5 μM S-25 and 0.5 μM Tol3 in buffers with a pH rangingfrom 3˜10. The buffers used were: 8 mM glycine-HCl, pH 3; 8 mM sodiumacetate, pH 4.4; 8 mM ammonium acetate, pH 6.0; 8 mM Tris-HCl, pH 7.4;40 mM Tris-HCl, pH 7.8; 8 mM Tris-HCl, pH 8.5; and 8 mM sodiumcarbonate, pH 10. The dye-protein mixture was incubated at roomtemperature (22° C.) for at least 15 minutes. Four replicates for eitherthe 50% aggregate or monomer at each pH were prepared and the plate wasscanned on a FLUOstar OPTIMA plate reader using an excitation wavelengthof 550 nm and an emission of 610 nm.

FIG. 3B shows the effect of pH on the linearity of aggregation specificfluorescence, as determined using 1.25 μM S-25 and 1.25 μM Tol3 in 50 mMof the following buffers: succinic acid-HCl, pH 5.0; histidine-HCl, pH7.0; and tris-HCl, pH 8.0. The total concentration of lysozyme was keptconstant at 20 but the percent of the total that was aggregated asopposed to monomeric was varied from 0% to 100% aggregate. At leastthree replicates of each sample was prepared, incubated at 22° in thedark for 15 minutes, then scanned on a FLUOstar OPTIMA plate readerusing an excitation wavelength of 550 nm and an emission of 610 nm.

Example 13. Linear Dynamic Range of Lysozyme Aggregate Detection Using aTwo Dye Combination ST (525& Tol3) Compared with Thioflavin T

Hen egg white lysozyme was solubilized in 10 mM HCl and heated to 65° C.for 90 hours to form aggregates. The signal from the aggregate wasdetermined after mixing aggregated lysozyme with monomeric lysozyme atdifferent ratios such that the total lysozyme concentration remained at20 μM protein. The readings were taken in 50 mM potassium phosphate, pH7, containing either ST (3 μM S-25 and 3 μM Tol3) or 5 μM thioflavin T.Protein was incubated with dye for 15 minutes prior to determining thefluorescence using a BioTek Synergy Mx plate scanner, with excitationsetting at 515 nm and emission setting at 603 nm, both with a 9 nmslit-width for S-25 and Tol3, and Thioflavin T was detected withexcitation setting at 435 nm and emission setting at 495 nm, both with a9 nm slit-width. Readings were taken in at least triplicate in a GreinerμClear black, clear bottom 96-well microplate. As seen in FIG. 4, theconcentration curve is more linear with S25/Tol3 as compared toThioflavin T.

Example 14. Effective Linear Dynamic Range of Antibody AggregateDetection Using a Two Dye Combination ST (525& Tol3), Compared withThioflavin T

Purified Rabbit anti-Goat IgG (4.26 mg/ml) was incubated in HCl, pH 2.7at 80° for 90 minutes to form aggregates. The signal from the aggregatewas determined after mixing aggregate with monomer at different ratiossuch that the total IgG concentration remained at 240 μg/ml protein. Thereadings were taken in 50 mM potassium phosphate, pH 7, containingeither ST (3 μM S-25 and 3 μM Tol3) or 5 μM thioflavin T. Protein wasincubated with dye for 15 minutes prior to determining the fluorescenceusing a BioTek SynergyMx plate scanner, with excitation setting at 515nm and emission setting at 603 nm, both with a 9 nm slit-width for S-25and Tol3, and thioflavin T was detected with excitation setting at 435nm and with emission setting at 495 nm, both with a 9 nm slit-width.Readings were taken in at least triplicate in a Greiner μClear black,clear bottom 96-well microplate. As can be seen in FIG. 5, the signalfrom ST is more than 10 times higher than the signal from thioflavin Tunder these conditions. Also the concentration curve is more linear withS-25/Tol3 as compared to thioflavin T.

Example 15. Protein Aggregate Detection as a Function of Protein Species

The linearity of aggregation induced fluorescence of S-25, Tol3 andThioflavin T (Thio-T) for four different proteins was determined. Theproteins were hen egg white lysozyme (results shown in FIG. 6A), rabbitanti-goat IgG (FIG. 6B), bovine insulin (FIG. 6C) and β-lactoglobulin(FIG. 6D)).

Chicken egg white lysozyme aggregate solution and monomer solution aswell as their mixtures were prepared as described in Example 12. Theprotein concentration was maintained at 20 μM, and the dye concentrationwas 2.5 μM in 50 mM Tris-HCl, pH 8. The ratio of aggregated protein tonative protein was varied from 0 to 100% aggregate. Each sample wasanalyzed in at least 3 replicates. The mixtures were incubated in thedark at 22° C. for 15 minutes, then the fluorescence intensity wasdetermined with a FLUOstar OPTIMA plate reader (BMG LABTECH) withexcitation setting at 550 nm and emission setting of 610 nm; while thefluorescence intensity for Thioflavin-T was determined using aSpectraMAX GeminiXS (Molecular Devices, with Softmax Pro 7.0) using anexcitation wavelength of 435 nm and emission wavelength of 495 nm.

Rabbit-anti-goat IgG (H&L, Pel-Freez®, formulated in the same manner asgoat-anti-mouse IgG, described in Example 2) was diluted to 29.4 μM withdouble deionized water adjusted to pH 2.7 using HCl. Then IgG aggregatewas prepared by shaking the protein solutions at 750 rpm in aThermomixer (Eppendorf) at 80° C. for 2 hours. Using a final proteinconcentration of 3 μM, the linearity of aggregation induced fluorescencewas determined as described above for lysozyme.

Insulin aggregate was prepared by dissolving bovine pancreas insulin(Sigma-Aldrich) at 170 μM in 100 mM HCl, which was subsequentlytransferred to a Thermomixer (Eppendorf), set at 750 rpm continuousshaking at 65° C. for 150 min. Using a final protein concentration of 20μM, the linearity of aggregation induced fluorescence was determined asdescribed above for lysozyme.

β-Lactoglobulin (BLG, Sigma-Aldrich) was dissolved at 1 mM in doubledeionized water. The aggregate was prepared by shaking the proteinsolutions at 750 rpm in a Thermomixer (Eppendorf) at 80° C., which wasstopped after 24 hours. Using a final protein concentration of 50 μM,the linearity of aggregation induced fluorescence was determined asdescribed above for lysozyme.

Example 16. Kinetics of Lysozyme Aggregation

A 1 mM solution of hen egg white lysozyme in 10 mM HCl was incubated at65° C. in an Eppendorf thermomixer shaking at 750 rpm. At the indicatedtimes, aliquots of the lysozyme were removed, diluted to 30 μM in 100 mMTris-HCl, pH 8.0, and incubated with 5 μM of the indicated dye. After 15minutes incubation, fluorescence intensity was determined with aFLUOstar OPTIMA plate reader (BMG LABTECH) at excitation wavelength of550 nm and emission wavelength of 610 nm; while the fluorescenceintensity for thioflavin-T was determined using a SpectraMAX GeminiXS(Molecular Devices, with Softmax Pro 7.0) using an excitation wavelengthof 435 nm and emission wavelength of 495 nm. Every sample was evaluatedin 4 replicates. As can be seen in FIG. 7, Tol3, S-25 and Thioflavin Tall detect similar kinetics for protein aggregate formation.

Example 17. Protein Aggregation as a Function of Temperature

A 0.9 mg/ml solution of goat-anti-mouse IgG (Pel Freeze) was prepared in73 mM sodium acetate, pH 4.5. This solution was incubated at 21° C. or50° C. for various times. After incubation, the solution was dilutedfurther to create a solution that was 50 mM histidine, pH 7, 0.45 mg/mlIgG, 2.5 μM S-25 and 2.5 μM Tol3. After 15 minutes further incubation,the fluorescence intensity was determined with a FLUOstar OPTIMA platereader (BMG LABTECH) at an excitation wavelength of 550 nm and emissionwavelength of 610 nm. As seen in FIG. 8, aggregation is much more rapidat 50° C. than at 21° C.

Example 18. Protein Aggregation as a Function of pH

Goat-anti-mouse IgG was diluted to 40 μM at either pH 7.6 in sodiumphosphate buffer, or adjusted to pH 2.46 using HCl. Both solutions werethen kept at 21° C. After various times, aliquots were removed anddiluted to a final concentration of 2 μM in 100 mM histidine buffer, pH7 with 2.5 μM S-25 and 2.5 μM Tol3. After 15 minutes further incubationat 21° C., the fluorescence intensity was recorded. As seen in FIG. 9,aggregation is observed to be much more rapid under acidic pHconditions.

Example 19. Illustration of High-Throughput Protein FormulationOptimization

(A). Goat anti-mouse IgG was diluted in sodium acetate, pH 4.5, thenmixed with the excipients shown in FIG. 10A giving a final concentrationof 400 mM sodium acetate, 18 μM IgG and the excipient concentrationshown in FIG. 10A. This mixture was heated to 50° C. for 6 hours. Afterthis incubation, the protein solution was diluted two-fold to give afinal concentration of 50 mM histidine buffer, originally pH 7, 2.5 μMS-25, 2.5 μM Tol3 and 9 μM IgG. After 30 minutes of incubation on theshaker, the fluorescence intensity was recorded on the plate reader(FLUOstar Optima). The fluorescence intensity from each individualexcipient was then compared with that without any excipient (value setas 1.0) as shown on the top of the corresponding excipient bar in FIG.10A.

(B). In the control plate, the IgG was added to the plate at the samevolume and concentration as in A. above, in 400 mM sodium acetate. Thismixture was heated to 50° C. for 6 hours, as described above. After 6hours, the excipient was added followed by S-25 and Tol3 to give all thefinal concentrations as in A. above. Similar to the sample plate, thefluorescence intensity from individual excipients was also compared withthat from water without any excipient (values set as 1.0) to obtain therelative fluorescent intensity as shown on the top of the correspondingexcipient bar in FIG. 10B.

(C). A ratio between the fluorescent intensity of the protein aggregatedwith the excipient versus the intensity derived from the proteinaggregated without excipient is a good measure of the effect of thegiven excipient on aggregation. FIG. 10C shows the ratio of fluorescenceintensity in the sample plate (A. above) divided by the fluorescenceintensity of the control plate (B. above). Those compounds with a valueof 1 (dotted line) do not significantly affect aggregation of IgG. Thosecompounds substantially higher than 1, such as 0.2% Triton X-100 induceaggregation of IgG. Those compounds with a value substantially lowerthan 1, such as 100 mM trehalose, inhibit or slow down aggregation ofIgG.

Example 20. Inhibition of Lysozyme Aggregation by Chitotriose

Hen egg white lysozyme (300 μM) was incubated with or withoutN,N′,N″-triacetyl-chitotriose (“Chitotriose”, 510 μM) in 10 mM potassiumphosphate, pH 7.3 for 16 hours. Aggregation was induced by 3.5 folddilution into 50 mM potassium phosphate, pH 12.2. Aggregation wasfollowed by removing an aliquot of the protein and diluting such thatthe final composition was 20 μM protein, 50 mM potassium phosphate, pH7, 3 μM S-25 and 3 μM Tol3. Protein was incubated with dye for 15minutes prior to determining the fluorescence using a BioTek Synergy Mxplate scanner, with excitation setting at 515 nm and emission setting at603 nm, both with a 9 nm slit-width. The zero time point was takenbefore dilution to pH 12.2. Readings were taken in at least triplicatein a Greiner μClear black, clear bottom 96-well microplate. Aggregationwas followed for several weeks at room temperature (19°-23° C.). As seenin FIG. 11, S-25 and Tol3 easily demonstrate that Chitotriose inhibitslysozyme aggregation, as previously demonstrated by Kumar et al. (2009).

Example 21. Thermal Shift Assays of BLG Aggregation

A solution containing 4 or 16 mg/mL of β-lactoglobulin (BLG) and2×SYPRO® Orange dye (Molecular Probes, supplied as 5000× with unknownconcentration) or 4 μM TOL3 or 4 μM S25 was prepared using 1×PBS, pH 7.4as the dilution buffer. This solution was then loaded into LightCycler®capillaries (20 μL, Roche Diagnostics GmbH). These capillaries were thenmounted on the sample holder of a LightCycler® 480 Real-Time PCR System(Roche), programmed to heat from 28° C. to 90° C. at 3° C./min, followedby cooling down to 28° C. at the same rate. The thermal shift curveswere achieved by plotting fluorescence intensity vs. temperature. Afterthe heating cycle, protein aggregates were visually apparent. However,SYPRO® Orange dye, known to detect protein, failed to show a meltingpeak, probably because of a low binding affinity to the aggregated BLG;but both TOL3 and S25 were able to detect BLG thermal shift peaks due tothe aggregation, as shown in FIG. 12. The temperature of aggregationdetected by TOL3 or S25 both showed a protein concentration dependence,down-shifting from 81˜83° C. to 71˜73° C. when the BLG concentration wasincreased from 4 mg/mL to 16 mg/mL, a characteristic of proteinaggregation, as opposed to protein unfolding. This demonstrates thatboth TOl3 and S25 are detecting aggregation thermal shift peaks of BLG,not transitions do to unfolding of the protein.

Example 22. Thermal Shift Assays of Carbonic Anhydrase II Aggregation

Carbonic anhydrase II (Sigma, 10 μM) containing 5×SYPRO® Orange or 10 μMTOL3 or S25 or Yat 2150 was prepared using either 50 mM sodium acetate,pH 4.5 or 25 mM PIPES, pH 7.0 buffer containing 100 mM NaCl and 0.5 mMEDTA. Sample preparation and the thermal shift assay were then performedusing the same conditions as described in Example 21. As shown in FIG.13, although SYPRO® orange and dyes of the invention all show thermalshift peaks, there is a ˜5° C. up-shift for peaks from dyes of theinvention, between pH 4.5 and pH 7.0. This also highlights thefundamentally different detection mechanism between SYPRO® Orange dyeand the dyes described in this invention; the former detects proteinunfolding, while the later detects protein aggregation.

Example 23. Comparison of Fluorescence Response Between Unfolded andAggregated Form of IgG Using Dyes of the Present Invention

(A) Chemical shift assay based on internal tryptophan fluorescence:Rabbit-anti-goat IgG (Pel Freeze) in 1×PBS buffer of pH 7.4 was mixedwith urea in 1×PBS to achieve a final IgG concentration of 0.25 mg/ml.After mixing on ice for 10 minutes, the fluorescence emission intensityat 330 nm was recorded by exciting at 280 nm using a MD-5020 fluorimeter(Phototechnology International). A chemical shift curve was plottedbased on internal tryptophan fluorescence intensity at each given ureaconcentration. Results are shown in FIG. 14A. Urea denatures proteinsbut prevents them from aggregating.

(B) A solution containing aggregated IgG (formed as in Example 15) ormonomeric IgG at 0.033 mg/mL, 4.55 M urea and 6.67 μM Tol3 was preparedand transferred into a microplate. After incubating at 4° C. degree forabout 10 minutes, the fluorescence was recorded. Two control solutionswithout IgG but with the same concentration of TOl3 were included, oneincluding 4.55M urea, another without urea. From the previous chemicalshift curve generated (FIG. 14A), 4.55 M urea is known to unfoldapproximately 60% of the IgG. The results shown in FIG. 14B indicatethat TOL3 is sensitive to IgG aggregates, which shows significantfluorescence enhancement relative to controls without IgG, but it is notsensitive to unfolded IgG monomer, which shows insignificantfluorescence enhancement relative to controls without IgG.

Example 24. PDI Isomerase Activity Assay by Monitoring InsulinAggregation Kinetics

(A) Turbidity assay: Protein disulfide isomerase (PDI, Assay Designs)was diluted with 0.5M of sodium phosphate, pH 6.8. A mixture was madewith insulin to give a final solution comprising 188 mM sodiumphosphate, pH 6.8, 5 mM Tris-HCl, 2 mM EDTA, 1 mM DTT, 1 mg/mL insulinand PDI at the desired concentrations (0, 5, 10, 15, 20, 25 μg/mL). Theoptical density (OD) at 630 nm was recorded immediately after theaddition of DTT in a 96-well microplate reader at 2 minute-intervals,with every well containing 300 μL solution. The OD from 0 μg/mL of PDIat any time point was used as a background value and was subtracted fromthe OD value of samples with PDI at the same time point. Results areseen in FIG. 15A.

(B) Fluorometric assay: PDI and insulin solutions were prepared as inthe turbidity assay described in (A) above. S25 and TOL3 were mixed withthe insulin solution and placed into a black Greiner flat bottom 96-wellplate. PDI solutions containing various amount of PDI were then added.Just prior to fluorescence recording, DTT was added. The final solutionwas 188 mM sodium phosphate pH 6.8, 5 mM Tris-HCl, 2 mM EDTA, 1 mM DTT,0.225 mg/mL insulin and PDI at 0, 5, 10, and 20 μg/ml. A FLUOstar Optimaplate reader was used to record the fluorescence change after 5 seconds'shaking with excitation set at 550 nm and emission set at 610 nm. Thefluorescence intensity from 0 μg/mL of PDI solution at the correspondingtime point was used as a background value and was subtracted from thecorresponding reading in the presence of enzyme. Results are seen inFIGS. 15B and C. The turbidity assay and fluorometric assay, though ofsignificantly different sensitivities, are orthogonal to each other,further supporting that dyes of the present invention monitoraggregation status and not unfolding status.

Example 25. Inhibition of β-Lactoglobulin Aggregation by HSP 27

Aggregation of β-lactoglobulin was monitored in the presence or absenceof the chaperone HSP 27. Aggregation of 8 mg/ml β-lactoglobulin wasmonitored using 1.25 μM Tol3 and 1.25 μM S25 in PBS, pH 7.4 with 2.5 mMEDTA and 0.05% sodium azide. When the chaperone HSP 27 was added it wasadded to a final concentration of 0.4 mg/ml. HSP 27 was also run in theabsence of β-lactoglobulin as a control. Aggregation was initiated byheating the protein solution to 68° C. in a 96 well half-volume clearplate (Biomol International, Inc). The fluorescence intensity was thenrecorded every 2 minutes, with shaking between reads. The excitationwavelength was set to 550 nm and the emission was set to 610 nm on a BMGFluorstar plate reader. The fluorescence intensity of the starting pointwas subtracted from the remaining points. The results (FIG. 16) indicatethat Hsp 27 can significantly prevent the aggregation of BLG at a massratio as low as 1:20. Since Hsp 27 is binding with unfolded BLGintermediate, thus preventing protein aggregation, the dyes aredetecting protein aggregation, as opposed to unfolding.

Other chaperone activity assays can be configured using β-lactoglobulinor other substrates, such as citrate synthase (CS). Table 6 showssuggestions for chaperone-to-CS ratios that should find application forthe disclosed assay methods.

TABLE 6 Chaperone:CS ratios. Chaperone system Members ADI catalog #sChaperone:CS DnaK/DnaJ/GrpE DnaK SPP-630 1:1 or less DnaJ SPP-640 GrpESPP-650 Hsp70/Hsp40 Hsp70 NSP-555, ESP-555, 1:1 or less SPP-758 Hdj1SPP-400 Hdj2 SPP-405 Mortalin SPP-828 Hsc70 SPP-751 Hsp70-A1 SPP-502,ESP-502 Hsp90 Hsp90 alpha SPP-776 Depends on Hsp90 beta SPP-777cochaperones Chaperonins Hsp60/Cpn10 NSP-540, ESP-540 1:1 or less(human) Cpn10 SPP-110 Chaperonins GroEL SPP-610 1:1 or less (bacterial)GroES SPP-620 Small heat shock Hsp25 SPP-510 20:01     proteins Hsp27SPP-715, SPP-716 Crystallins SPP-225, SPP-226, SPP-235, SPP-236 ERchaperones Grp78 SPP-765 5:1     PDI SPP-891 10:1     Erp72H00009601-Q01 20:1     (abnova) Grp94 SPP-766 Depends on (ER Hsp90)cochaperones Nascent chain NAC none 20:1     chaperones Trigger Factornone 20:1    

Chaperone: CS ratios are based upon the known biology of the individualsystems. Active folders are likely to show significant signal at lessthan 1:1 molar ratio to substrate, as each chaperone complement will beable to inhibit aggregation while it actively folds. Aggregateinhibitors like the small heat shocks and trigger factor requiresubstantially more, as they need to saturate the solution to preventaggregation. Pairs of holders and folders (e.g., crystalline with lowHsp70 complex) may provide synergistic effects.

Example 26. Monitoring Protein Stability in an Agitated Solution

One method of creating aggregated proteins is by agitation of theprotein solution. Goat-anti-mouse IgG (12.8 mg/mL, Pel-freezeBiologicals) was supplied in 10 mM sodium phosphate, 150 mM NaCl, 0.05%sodium azide, pH 7.2, filtered through 0.2 μm filter. The stirringexperiment was performed by stirring 200 μL of IgG solution as suppliedat 22° C. in a 4 mL amber glass vial with flat bottom at 990 rpm usingVariomag® Poly electronic stirrers. The control (without stirring) wasalso kept at 22° C. The stirring bar was 1×0.4×0.2 cm³.

A BioTek plate reader with a filter set as 550 (excitation)/603 nm(emission) and 9 nm filter band on both excitation and emission was usedto scan from the bottom of the plate. 5 μL of the IgG solution atvarious time points (stirred or non-stirred) was added into 95 μL of 2.5μM TOL3, 2.5 μM of S25 and 50 mM potassium phosphate, pH 7.0 andincubated for 20 minutes. Every time point was replicated twice. Thefluorescence of free dye was subtracted from that of the IgG/dye mixingsolution for both the stirred sample and non-stirred control. Theresults (FIG. 17) indicate that the TOL3/S25 dye mix can detectagitation induced aggregation.

Example 27. Thermal Shift Assay to Find Thermally Stabilizing Buffersfor DNA Polymerase I Klenow Fragment

In molecular biology, enzymes are often required that function atelevated temperatures. Enzymes produced by mesophilic organisms usuallydenature at elevated temperatures, followed by aggregate formation. Arapid fluorescence-based assay was developed for assessing a range ofparameters impacting the thermal stability of an enzyme. Overall,protein stability was monitored by a fluorogenic dye that selectivelydetects aggregated protein. Stability can be measured in the presence ofdifferent buffers, cryoprotectants and excipients. By systematicallyraising the temperature of the protein in solution, the temperature atwhich the protein aggregates (T_(agg)) can be determined. Using thismethod with Klenow DNA polymerase, it was determined that trehalosesignificantly increases T_(agg). The DNA polymerase activity of theenzyme is significantly enhanced at 50° C. in the presence of trehaloseunder the same conditions. Low amounts of the detergents Tween20® andTriton X-100® (0.05%) decreased T_(agg) and also compromised enzymeactivity, especially at elevated temperatures. The assay facilitatesscreening for buffers and additives that structurally stabilize aprotein of interest at elevated temperatures.

Fifty units of DNA polymerase I Klenow fragment (New England Biolabs,Ipswich, Mass.) was incubated in several different buffers andexcipients in the presence of 2.5 μM YAT2150 dye. The temperature wasslowly raised and the fluorescence was determined using a Qiagen(Valencia, Calif.) Rotorgene real-time thermocycler with an excitationfilter at 530 nm and an emission filter of 610 nm. Table 7 shows thedetected aggregation temperatures in Buffer 1 (10 mM bis-Tris propane,10 mM MgCl₂, 1 mM dithiothreitol, pH 7), Buffer 2 (50 mM NaCl, 10 mMTris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.9), Buffer 3 (100 mMNaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.9), Buffer4 (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate,1 mM dithiothreitol, pH 7.9), Buffer 2 with 0.5 M trehalose, Buffer 2with 0.05% Tween20® and Buffer 2 with 0.05% Triton X-100®.

TABLE 7 Effect of various buffers on aggregation temperature of DNApolymerase I Klenow fragment. Buffer T_(agg) Buffer #1 59.1° +/−0.3°Buffer #2 59.7° +/−0.2° Buffer #3 60.2° +/−0.3° Buffer #4 59.8° +/−0.4°Buffer #2 Trehalose 62.6° +/−0.3° Buffer #2 Tween 20 58.4° +/−0.3°Buffer #2 Triton X-100 57.8° +/−0.2°

This data demonstrates that trehalose significantly raises theaggregation temperature in Buffer 2. To show if this extends to enzymeactivity, the DNA polymerase was incubated in Buffer 2 with 400 μM dATP,dCTP, dGTP and fluorescein-12-dUTP at 37° C., 42° C. and 50° C. After 15minutes at the given temperature, the DNA template and primer were addedfor an additional 15 minutes at the given temperature. The template usedwas 5-ACTTCTTACT TCTTACTTCT TACTTCTTAC TTCTTACTTC TTACTTCTTA CTTCTTACTTCTTCATTGGT CATCTCGATC CATGACCTCA GC-3′ and the primer was 5′-TTGCTGAGGTCATGGATCGA GA-3′. The amount of oligo extended full length is shown inTable 8, as measured by relative fluorescence of the incorporatedfluorescein.

TABLE 8 Oligonucleotide extension by DNA polymerase I Klenow fragmentusing various buffer additives during incubation at three temperatures.Additive in Buffer 2 37° 42° 50° None 0.507 0.303 0.189 0.5M Trehalose0.567 0.449 0.308 0.05% Tween20 0.317 0.154 0.180 0.05% Triton X-1000.257 0.159 0.170This data shows a correlation with the increased aggregation temperaturein the presence of 0.5 M trehalose and the enzyme activity at elevatedtemperatures.

Example 28. YAT2150 Dye Detecting Aggregated Proteins within Fixed andPermeabilized Cells

In mammalian cells, aggregated proteins may be concentrated bymicrotubule-dependent retrograde transport to perinuclear sites ofaggregate deposition, referred to as aggresomes. Aggresomes areinclusion bodies that form when the ubiquitin-proteasome machinery isoverwhelmed with aggregation-prone proteins. Typically, an aggresomeforms in response to some cellular stress, such as hyperthermia, viralinfection, or exposure to reactive oxygen species. Aggresomes appear toprovide a cytoprotective function by sequestering the toxic, aggregatedproteins and may also facilitate their ultimate elimination from cellsby autophagy. Certain cellular inclusion bodies associated with humandisease are thought to arise from an aggresomal response, including Lewybodies associated with neurons in Parkinson's disease, Mallory bodiesassociated with liver cells in alcoholic liver disease, and hyalineinclusion bodies associated with astrocytes in amyotrophic lateralsclerosis.

The ability of YAT2150 to detect aggregated proteins within fixed andpermeabilized cells was evaluated. Human cervical adenocarcinomaepithelial cell line HeLa was obtained from American Type CultureCollection (ATCC, Manassas, Va.). HeLa cells were routinely cultured inEagle's Minimum Essential Medium (ATCC) with low glucose, supplementedwith 10% fetal bovine serum (FBS) (ATCC) and 100 U/ml penicillin with100 μg/ml streptomycin (Sigma-Aldrich). Cells were maintained in asaturated, humidified atmosphere at 37° C., 5% CO₂ and 95% air. HeLacells were grown on glass slides or polystyrene tissue culture dishesuntil ˜80% confluent. The cells were treated with various modulators orvehicle at various concentrations and time intervals, as detailed inTable 9. Proteasome inhibitors MG-132 (Enzo Life Sciences Inc.),lactacystin (Enzo Life Sciences Inc.), bortezomib (Velcade®) (SelleckChemicals LLC, Houston, Tex.) and epoxomicin (Enzo Life Sciences Inc.)were employed in the studies. The histone deacetylase 6 inhibitorN-hydroxy-7-[5-(4-tertbutoxycarbonylaminophenyl)-3-isoxazolecarboxamido]heptamide (BML-281) was also obtained from Enzo Life Sciences Inc.Negative control cells were treated with a vehicle (DMSO, media or othersolvent used to reconstitute or dilute the inducer or inhibitor) for anequal length of time under similar conditions. The cells weresubsequently washed with PBS, and fixed in 4% formaldehyde in PBS for 30min at room temperature, then permeabilized with 0.5% Triton X-100, 3 mMEDTA in PBS on ice, for 30 minutes. The cells were washed with PBS, andthen 500 nM of YAT2150 dye was added. The samples were incubated for 30minutes at room temperature, protected from light. The cells were washedwith PBS, covered with glass coverslips and observed using afluorescence microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany)equipped with a Texas Red filter set. Images were acquired with a 63×objective lens (Carl Zeiss, Inc).

μM Induction Cell Aggresome Treatment Target Effect used Time (hrs) LineFormation Starvation Inhibits mammalian Activates N/A 1~4 HeLa, Notarget of rapamycin autophagy Jurkat (mTOR) Rapamycin Inhibits mammalianActivates 0.2 6~18 HeLa, No target of rapamycin autophagy Jurkat (mTOR)PP242 ATP-competitive Activates 1 18 Hela No inhibitor of mTOR autophagyLithium Inhibits IMPase and Activates 10,000 18 HeLa, No reduce inositoland autophagy Jurkat IP₃ levels; mTOR- independent Trehalose Unknown,mTOR- Activates 50,000  6 HeLa, No independent autophagy JurkatBafilomycin A1 Inhibits Vacuolar- Inhibits 6~9 18 HeLa, Yes ATPaseautophagy ×10⁻³ Jurkat Chloroquine Alkalinizes Inhibits 10~50 18 HeLa,Yes Lysosomal pH autophagy Jurkat Tamoxifen Increases the Activates 4~106~18 HeLa, Yes intracellular level of autophagy Jurkat ceramide andabolishes the inhibitory effect of PI3K Verapamil Ca²⁺ channel blocker;Activates 40-100 18 HeLa, Yes reducesintracytosolic autophagy JurkatCa²⁺ levels; mTOR- independent Hydroxy- Alkalinizes Inhibits 10 18 HeLa,Yes chloroquine Lysosomal pH autophagy Jurkat Loperamide Ca²⁺ channelActivates 5 18 HeLa No blocker;reduces intra- autophagy cytosolic Ca²⁺levels; mTOR-independent Clonidine Imidazoline-1 Activates 100 18 HeLaNo receptor agonist; autophagy reduces cAMP levels; mTOR-independentMG-132 Selective proteasome Induce 2~5 18 HeLa, Yes inhibitor aggresomeJurkat Epoxomicin Selective proteasome Induce 0.5 18 HeLa Yes inhibitoraggresome Lactacystin Selective proteasome Induce 4 Yes inhibitoraggresome Velcade ® Selective proteasome Induce 0.5 18 HeLa Yesinhibitor aggresome amyloid beta Induce oxidative Induce 25 18 SK-N- Yespeptide 1-42 stress aggresome SH Norclomipramine Alkalinizes Inhibits5~20 18 HeLa Yes Lysosomal pH autophagy

MG-132, a relatively nonspecific proteasome inhibitor, has also beenshown to perturb protein homeostasis, inducing both the unfolded proteinresponse (UPR) and the heat shock response (HSR) (Mu et al., 2008;Murakawa et al., 2007). MG-132 is known to accelerate the formation ofperinuclear aggresomes as well as inclusion bodies within cells(Beaudoin S et al., 2008). After treating cells with MG-132, YAT2150 dyewas found to readily highlight aggregated protein cargo accumulatingwithin vacuolar cytoplasmic structures, as observed by fluorescencemicroscopy (FIG. 18). Examination of the distribution of the fluorescentdye within cells treated with MG-132, revealed a punctate pattern ofcytoplasmic staining, as well as staining of certain inclusion bodieswithin or immediately adjacent to the nucleus itself. Multiplecytoplasmic inclusion bodies were readily discerned using thefluorescent dye. It should be noted that true aggresomes arecharacterized by a single large protein aggregate in cells thatco-localizes with the centrosome in a microtubule-dependent fashion. Theformation of the multiplicity of inclusion bodies observed upon MG-132treatment was not sensitive to the histone deacetylase 6 inhibitor,N-hydroxy-7-[5-(4-tertbutoxycarbonylaminophenyl)-3-isoxazolecarboxamido]heptamide(BML-281) (Kawaguchi et al., 2003) or nocodazole (data not shown). Thissuggests that the generated inclusion bodies do not meet the strictestdefinition for aggresomes. The described probe appears to detectaggregated protein cargo within a variety of inclusion bodies,regardless of whether they are co-localized with the centrosome orformed in a microtubule-dependent manner.

The ability to detect aggresomes and related inclusion bodies wasfurther demonstrated using various potent, cell permeable, and selectiveproteasome inhibitors: lactacystin, epoxomicin and bortezomib(Velcade®), as shown in FIG. 19. Previous studies have shown thatbortezomib-mediated proteasomal inhibition results in the accumulationof large quantities of ubiquitin-conjugated proteins organized intoperinuclear structures termed “aggresomes” (Nawrocki et al., 2006). Allof the tested proteasome inhibitors induced the accumulation ofcytoplasmic inclusion bodies within the cells, as demonstrated with theYAT2150 dye. Efforts to stain inclusion bodies in living cells were notmet with success, however. Instead of discrete punctuate staining ofaggregated cargo, a weak, diffuse cytoplasmic staining was observed thatdiffered little between control cells and cells treated with aproteasome inhibitor. This could possibly be due to poor access of thedye to the contents of membrane-bound vacuolar structures within thecells.

Example 29. Co-Localization of Aggregated Protein with Ubiquitinylationand Various Pathway Proteins Implicated in Autophagy

Antibodies were obtained from the following commercial sources:fluorescein-labeled p62 and LC3 reactive rabbit polyclonal antibodiesand ubiquitin-reactive mouse monoclonal antibody (clone EX-9) wereobtained from Enzo Life Sciences, Ltd. (Exeter, UK). These labeledconjugates were produced by direct labeling of antibodies raised top62-derived, LC3-derived, and ubiquitin-derived peptides, respectively.A mouse monoclonal antibody reactive with human tau (clone tau-13)(Covance Inc, Emeryville, Calif.) is able to stain brain tissue early inAlzheimer's disease. It was used in conjunction with Alexa Fluor® 488dye-labeled goat anti-mouse secondary antibody from Life Technologies(Carlsbad, Calif.). Alexa Fluor® 488 dye-labeled beta amyloid reactivemouse monoclonal antibody (clone 6E10), which is specifically reactiveto amino acid residues 1-16 of the human β-amyloid peptide, was obtainedfrom Covance Inc.

For antibody co-localization studies: cells were treated overnight with5 μM MG-132, then fixed and permeabilized using the protocol in Example28. The cells were then incubated in PBS containing 3% bovine serumalbumin (blocking buffer). Fluorescein-labeled p62, LC3 and ubiquitin(clone EX-9) reactive antibodies were diluted to a concentration of 1μg/mL in blocking buffer and incubated for 1 h at room temperature.Cells were then washed in PBS containing 0.1% Tween-20 for 15 min. Next,the cells were stained with YAT2150 dye for 30 minutes at roomtemperature and washed with PBS, covered with glass cover slip, sealedwith nail polish, and observed by fluorescence microscopy using a TexasRed filter set for the YAT2150 dye, and an FITC filter set forfluorescein-labeled antibodies, respectively. All images were acquiredwith a 63× objective lens (Carl Zeiss, Inc).

Co-localization of fluorescently-labeled ubiquitin antibody conjugatewith YAT2150 dye is shown in FIG. 20A, highlighting interactions betweenaggregated protein cargo and ubiquitinylation status. The ubiquitinsignal is observed to be co-localized exclusively with the aggregatedprotein cargo, but it should be remembered that cells were fixed andpermeabilized, which likely removed any free ubiquitin andubiquitinylated substrates present in the cytosol. FIG. 20B demonstratesthat a fluorescein-conjugated antibody directed towards p62 (aubiquitin-binding scaffold protein that co-localizes withubiquitinylated protein aggregates) also co-localizes with the YAT2150dye within cells treated with MG-132. Furthermore, co-localizationbetween fluorescein-labeled antibodies reactive with LC3 (aubiquitin-like autophagy cascade protein residing in the phagophoremembrane) and aggregated protein cargo was demonstrated, as shown inFIG. 20C. The described co-localization studies demonstrate thecapability of the YAT2150 dye to be used to analyze the interactionsbetween aggregated protein cargo, protein post-translationalmodifications, and various autophagy pathway proteins. The longwavelength red emission of the fluorescent probe is especially suitablefor studies using green fluorescent dye conjugates, such as fluorescein,Alexa Fluor® 488, Oregon Green® 488, BODIPY®-FL, HiLyte Fluor™ 488 andDyLight® 488.

Example 30. Cell Culture-Based Model Mimicking Elements of Alzheimer'sDisease Pathology

The human SK-N-SH neuroblastoma cell line was obtained from AmericanType Culture Collection (ATCC, Manassas, Va.). SK-N-SH cells wereroutinely cultured in Eagle's Minimum Essential Medium (ATCC) with lowglucose, supplemented with 10% fetal bovine serum (FBS) (ATCC) and 100U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich). Amyloid betapeptide 1-42 (21^(st) Century Biochemicals, Marlboro, Mass.) was addedto the culture medium and SKN-SH cells were incubated overnight toinduce aggresome formation. SMER28 (Enzo Life Sciences Inc.), an inducerof autophagy, was employed to block this accumulation A cellculture-based assay mimicking the accumulation of β-amyloid, as observedin Alzheimer's disease, was established.

FIG. 21B shows YAT2150 dye is able to detect amyloid fibrils within anSK-N-SH human neuroblastoma cell line induced to form inclusion bodiesby overnight incubation with exogenously added amyloid beta 1-42peptide. Furthermore, SMER28, a small molecule inducer of autophagy wasevaluated with respect to its effect on β-amyloid peptide accumulationwithin the cells. SMER28 has previously been shown to act via anmTOR-independent mechanism to increase autophagosome synthesis andenhance the clearance of model autophagy substrates, such as[A53T]α-synuclein and mutant huntingtin fragments (Sarkar et al., 2007;Renna et al., 2010). It has also been demonstrated that SMER28attenuates mutant huntingtin-fragment toxicity in Drosophila models,suggesting therapeutic potential. As shown in FIGS. 21C & D, SMER28 wasable to substantially reduce accumulation of β-amyloid peptide inSK-N-SH human neuroblastoma cells, suggesting this assay couldpotentially enable screening of aggregation inhibitors relevant toneurodegenerative disease, in an authentic cellular context.

Example 31. Detecting Protein Aggregates in Post-Mortem Brain TissueSections from Patients with Alzheimer's Disease

Post-mortem brain tissue (cerebellum) from patients with Alzheimer'sdisease and human adult normal brain tissue (cerebellum) were obtainedfrom BioChain Institute, Inc. (Hayward, Calif.). All tissue samples werereceived from certified tissue vendors who guarantee that they werecollected with informed consent from the donors and their relatives, allsamples were excised by licensed physicians, all normal and diseasedtissues were determined by the donor's clinical reports and allcollections were made with the relevant requirements for ethicscommittee/IRB approvals. The frozen tissue sections were 5-10 μm inthickness, mounted on positively charged glass slides, and fixed withcold acetone by the manufacturer. The embedded tissue sections werefixed in formalin immediately after excision, and embedded in paraffin.Tissue sections were ˜5 μm in thickness, and mounted on positivelycharged glass slides by the manufacturer.

Paraffin-embedded tissue sections were deparaffinized prior to staining.Briefly, the microscope slide-mounted specimen was immersed in a xylenesubstitute bath until the paraffin was solubilized. The deparaffinizedspecimens were then washed with a series of alcohol solutions ofdecreasing alcohol concentration, to remove xylene, before a final washwith water. The tissue sections were then fixed with 4% formaldehyde inPBS for 15 min at 37° C. Following washing in deionized water, tissuesections were stained with either 1 μM thioflavin T in PBS or 500 nMYAT2150 dye for 3 min, rinsed in water and destained in 1% acetic acidfor 20 min. Finally the tissues sections were washed thoroughly inwater, dehydrated, covered with glass coverslips, mounted in anti-fademounting medium and observed using a fluorescence microscope (CarlZeiss, Inc.) with an FITC filter set for thioflavin T and a Texas Redfilter set for YAT2150 dye, respectively. All images were acquired witha 63× objective lens (Carl Zeiss, Inc).

For the antibody co-localization studies, tissue sections were stainedwith YAT2150 dye as described above. The tissue sections were thenblocked in PBS containing 3% bovine serum albumin (blocking buffer).Tau-reactive monoclonal antibody (clone tau-13) and Alexa Fluor® 488labeled beta amyloid reactive monoclonal antibody (clone 6E10) werediluted to a concentration of 2 μg/mL in blocking buffer and incubatedfor 1 h at room temperature. Tissues were then washed in PBS containing0.1% Tween-20 for 15 min. For tissues incubated with Tau-13 antibody,the slides were subsequently incubated with Alexa Fluor® 488 goatanti-mouse secondary antibody for 30 min at room temperature. Finallythe tissue sections were washed with PBS, covered with glass coverslips,mounted in anti-fade mounting medium and observed using a fluorescencemicroscope (Carl Zeiss, Inc.) with a Texas Red filter set for YAT2150dye and FITC filter set for labeled antibodies, respectively. All imageswere acquired with a 63× objective lens (Carl Zeiss, Inc.).

Thioflavin T (ThT) is a widely employed histological probe for detectingthe formation of amyloid fibrils in brain tissue (Gunilla et al., 1999).However, this dye is not an ideal predictor of the degree offibrillization because its fluorescence varies substantially dependingupon the structure and morphology of the amyloid fibrils. It was foundthat the dye generates fairly high background and weak fluorescentsignal in brain tissue sections, as shown in FIG. 22A. Thus, optimizedprotocols for the detection of amyloid plaques in frozen andparaffin-embedded tissue sections of human brain were developed usingthe YAT2150 dye. Relative to ThT, this novel probe demonstratessignificantly higher fluorescence emission intensity enhancement in thepresence of amyloid protein fibrils and low non-specific background(shown in FIG. 22B). In addition, use of antibodies directed againstβ-amyloid and tau protein, in conjunction with the YAT2150 dye, confirmthe selectivity of the probe for detection of amyloid plaques inpost-mortem brain tissue of patients with Alzheimer's disease (FIGS. 22A& B).

Example 32. Utilizing Flow Cytometry to Quantify the Accumulation ofProtein Aggregates within Cells

Human leukemic Jurkat cells were obtained from ATCC. Jurkat cells weregrown in suspension in RPMI medium supplemented with 10% (v/v) FBS,penicillin (100 U/ml), streptomycin (100 μg/ml), and glutamine (200 mM).Jurkat cells were maintained in a saturated, humidified atmosphere at37° C., 5% CO₂ and 95% air.

Jurkat cells were grown to log phase, and treated with 5 μM MG-132 orwith vehicle for 16 hours. At the end of the treatment, adherent cellswere trypsinized; while Jurkat cells were simply collected bycentrifugation (400×g for 5 min). Samples were resuspended at 1×10⁶ to2×10⁶ cells per ml. For each group, triplicate samples were prepared.The cells were washed with PBS, fixed in 4% formaldehyde in PBS for 30min and then permeabilized with 0.5% Triton X-100, 3 mM EDTA, pH 8 onice, for 30 minutes. The cells were then washed, and resuspended in 500μL of 200 nM YAT2150 dye. The samples were incubated for 30 minutes atroom temperature, protected from light. Experiments were performed usinga FACS Calibur benchtop flow cytometer (BD Biosciences, San Jose,Calif.) equipped with a blue (488 nm) laser. YAT2150 dye fluorescencewas measured in the FL3 channel. No washing was required prior to theflow cytometric analysis.

For the immunocytochemistry study, after fixing and permeabilizing thecells, the cells were blocked in PBS containing 3% bovine serum albuminfor one hour. Fluorescein-labeled p62 antibody was diluted to aconcentration of 2 μg/mL in blocking buffer and incubated with the cellsfor 1 h at room temperature. Cells were then washed in PBS containing0.1% Tween-20 for 15 min. Data was acquired by FACS Calibur benchtopflow cytometer (BD Biosciences, San Jose, Calif.) equipped with a blue(488 nm) laser, with the antibody signal measured in the FL1 channel.

All of the experiments were performed at least three times. Flowcytometry data were analyzed by comparison of mean fluorescence, throughcalculation of a term we refer to as the Aggregation Propensity Factor(APF), having the following definition.

APF=100×((MFI_(treated)-MFI_(control))/MFI_(treated)), whereinMFI_(treated) and MFI_(control) are the mean fluorescence intensityvalues from control and treated samples.This metric is based upon a similar approach that is commonly employedin the assessment of fluorescent signal between control and treatedgroups in multidrug resistance experiments, using a term referred to asMultidrug Resistance Activity Factor (MAF) (Hollo et al., 1994). APF isa unitless term measured as the difference between the amount of theYAT2150 dye accumulated within cells in the presence and absence of aproteasome inhibitor or other inducer of aggresome or inclusion bodyformation or protein aggregation. The fluorescence measurement in thepresence of the proteasome inhibitor constitutes the maximal potentialfluorescence for the given cell population when aggregated protein cargohas been generated. This represents a standardization method, whicheliminates unknown cell type-specific variables that might influence dyeaccumulation, such as cell size, shape and volume, allowing thepotential for intra- and inter-laboratory comparison of test results andAPF values.

A flow cytometry cell-based assay was next developed using the YAT2150dye. FIG. 24A demonstrates typical results of flow cytometry-basedanalysis of cell populations using the YAT2150 dye. Uninduced controland 5 μM MG-132-treated Jurkat cells were employed in the investigation.After 16 hours treatment, fixed and permeabilized cells were stainedwith the YAT2150 dye and then analyzed without washing by flowcytometry. Results are presented using histogram overlay graphs. Controlcells displayed minimal fluorescence staining with the dye. The YAT2150dye signal increased about three-fold in the MG-132 treated cells,readily demonstrating that MG-132 induced protein aggregate formation inJurkat cells. An APF value of approximately 72, as defined above,demonstrates that the control and treated cell populations were readilydistinguishable by flow cytometry. For comparison, an MAF cut-off valueof about 20-25 is routinely employed in flow cytometry assays ofmulti-drug resistance (Hollo et al., 1994). Protein aggregateaccumulation in the Jurkat cells was confirmed by flow cytometryanalysis using fluorescein-conjugated p62-reactive antibody (FIG. 24B).Thus, the described assay allows, for the first time, easyquantification of aggresome accumulation by flow cytometry. Theadvantage of the dye-based approach relative to the antibody one is thatstaining and analysis are much more rapid. Simultaneous staining withthe fluorescein conjugated antibody and the red fluorescent dye was alsofeasible (data not shown).

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In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A compound comprising the structure

wherein m and n are independently 1, 2 or 3; wherein L is a linker armcomprising carbon, sulfur, oxygen, nitrogen, or any combination thereof;wherein R₁, R₂, R₃, R₄, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₉, R₂₀,R₂₁ and R₂₂ are independently hydrogen, halogen, amino, ammonium, nitro,sulfo, sulfonamide, carboxy, ester, cyano, phenyl, benzyl, an alkylgroup wherein the alkyl group is saturated or unsaturated, linear orbranched, substituted or unsubstituted, an alkoxy group wherein thealkoxy group is saturated or unsaturated, branched or linear,substituted or unsubstituted, or when taken in combination R₁ and R₂, orR₃ and R₄, or R₉ and R₁₀, or R₁₁ and R₁₂, or R₁₃ and R₁₄, or R₁₅ andR₁₆, or R₁₉ and R₂₀, or R₂₁ and R₂₂ form a five or six membered ringwherein the ring is saturated or unsaturated, substituted orunsubstituted, and wherein R₉ and R₁₀, or R₁₁ and R₁₂, or R₁₃ and R₁₄,or R₁₅ and R₁₆ can comprise alkyl chains that are joined together,wherein a quinoline moiety can be formed; wherein R₇, R₈, R₁₇ and R₁₈are independently hydrogen, Z, an alkyl group wherein the alkyl group issaturated or unsaturated, linear or branched, substituted orunsubstituted, an alkoxy group wherein the alkoxy group is saturated orunsaturated, branched or linear, substituted or unsubstituted, or whentaken together, R₇ and R₈ and R₁₇ and R₁₈, may form a 5 or 6 memberedring wherein the ring is saturated or unsaturated, substituted orunsubstituted; wherein Z comprises a carboxyl group (CO₂ ⁻), a carbonateester (COER₂₅), a sulfonate (SO₃ ⁻), a sulfonate ester (SO₂ER₂₅), asulfoxide (SOR₂₅), a sulfone (SO₂CR₂₅R₂₆R₂₇), a sulfonamide(SO2NR₂₅R₂₆), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ⁻ER₂₅), aphosphate diester (PO₂ER₂₅ER₂₆), a phosphonate (PO₃ ⁼) a phosphonatemonoester (PO₂ ⁻ER₂₅) a phosphonate diester (POER₂₅ER₂₆), athiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ⁻ER₂₅) athiophosphate diester (PSOER₂₅ER₂₆), a thiophosphonate (PSO₂ ⁼), athiophosphonate monoester (PSO⁻ER₂₅) a thiophosphonate diester(PSER₂₅ER₂₆), a phosphonamide (PONR₂₅R₂₆NR₂₈R₂₉), its thioanalogue(PSNR₂₅R₂₆NR₂₈R₂₉), a phosphoramide (PONR₂₅R₂₆NR₂₇NR₂₈R₂₉), itsthioanalogue (PSNR₂₅R₂₆NR₂₇NR₂₈R₂₉), a phosphoramidite (PO₂R₂₅NR₂₈R₂₉)or its thioanalogue (POSR₂₅NR₂₈R₂₉) wherein E is independently O or S;wherein R₂₅, R₂₆, R₂₇, R₂₈, and R₂₉ are independently a hydrogen, anunsubstituted straight-chain, branched or cyclic alkyl, alkenyl oralkynyl group, a substituted straight-chain, branched or cyclic alkyl,alkenyl or alkynyl group wherein one or more C, CH or CH₂ groups aresubstituted with an O atom, N atom, S atom, or NH group, or anunsubstituted or substituted aromatic group; wherein Z is attacheddirectly, or indirectly through a second linker arm comprising carbon,sulfur, oxygen, nitrogen, and any combinations thereof and wherein thesecond linker arm may be saturated or unsaturated, linear or branched,substituted or unsubstituted or any combinations thereof; wherein R₅,R₆, R₂₃ and R₂₄ can independently be hydrogen or an alkyl group whereinthe alkyl group is saturated or unsaturated, linear or branched,substituted or unsubstituted, or when taken in combination R₅ and R₆ orR₂ and R₅ or R₃ and R₆ or R₂₃ and R₂₄ or R₂₂ and R₂₃ or R₂₀ and R₂₄ forma five or six membered ring wherein the ring is saturated orunsaturated, substituted or unsubstituted; and wherein said compound ismodified to comprise a reactive group which is an isocyanate,isothiocyanate, monochlorotriazine, dichlorotriazine,4,6,-dichloro-1,3,5-triazine, mono- or di-halogen substituted pyridine,mono- or di-halogen substituted diazine, maleimide, haloacetamide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol,azide, 3-(2-pyridyl dithio)-propionamide, glyoxal or aldehyde group. 2.The compound of claim 1, wherein the compound exhibits increasedfluorescence in the presence of an aggregated form of a protein whencompared to the fluorescence exhibited when the compound is in thepresence of the unaggregated form of the protein.
 3. The compound ofclaim 1, comprising the structure

modified to comprise the reactive group, or

modified to comprise the reactive group.
 4. The compound of claim 4,wherein each of R₅, R₆, R₂₃ and R₂₄ are a methyl or an ethyl moiety. 5.The compound of claim 1, wherein the compound is S25, S43, TOL3,YAT2134, YAT2148, YAT2149, S13, YAT2135, YAT2324 or YAT2150, modified tocomprise the reactive group.